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Chapter 2
233
41 Introduction
In recent years a number of compounds previously considered non-
degradable are also being degraded by microorganisms suggesting that
under selective pressure of environmental pollution microbes develop the
ability to degrade recalcitrant xenobiotics However the fact that many
pollutants are still persistent in the environment reflects the inadequacy of the
current microbial catabolic capacity to deal with such pollutants That is the
kinetics of process may be much slower than desired This has stimulated
the development of bioremediation technologies which may be applied to
many different environmental situations One of the new developing
technologies involves the genetic engineering of natural microorganisms with
enhanced or new degradative capabilities for bioaugmentation of selected
contaminated environments Molecular biology offers the tools to optimize
the biodegradative capacities of microorganisms accelerate the evolution of
new activities and construct totally new pathways through the assemblage
of catabolic segments from different microbes Although the number of
genetically engineered microbes (GEMs) for potential use in biodegradation is
not large these recombinant microbes function in microcosms according to
their design The survival and fate of recombinant microbes in different
ecological niches under laboratory conditions is similar to what has been
observed for the unmodified parental strains rDNA both on plasmids and on
the host chromosome is usually stably inherited by GEMs The potential
lateral transfer of rDNA from the GEMs to other microbes is significantly
diminished though not totally inhibited when rDNA is incorporated on the
host chromosome The behaviour and fate of GEMs can be predicted more
accurately through the coupling of regulatory circuits that control the
expression of catabolic pathways to killing genes so that the GEMs survive in
polluted environments but die when the target chemical is eliminated (Ramos
et al 1994)
The formation of DDD from DDT is also a common reaction among soil
microorganisms (Guenzi and Beard 1968) Chacko et al (1966) isolated
numerous actinomycetes (Nocardia sp Streptomyces aureofaciens
Streptomyces cinnamoneus Streptomyces viridochromogenes) from soil
Chapter 2
234
which readily degraded DDT to DDD Wedemeyer (1967) studied DDT
metabolic pathway by incubation of proposed intermediates with organisms
and examining the products formed The metabolism of DDT in Aerobacter
aerogenes goes in order DDT DDD DDMU DDNU DDOH DDA
DBP and direct conversion of DDT to DDE (Wedemeyer 1967) Reports
on the involvement of enzymes in the degradation of DDT indicate the
presence of enzymes like dioxygenases (Nadeau et al 1994)
dehydrogenases (Bourquin 1977) oxygenases (Ahmed et al 1991) Only a
few enzymes have been described in DDT degradative pathway (Singh et al
1999) The conversion of DDT to DDD involves a dehydrochlorination step
In our laboratory an attempt was made to clone sequence and express the
gene responsible for this step
42 Materials
Luria- Bertoni broth and ampicillin were procured from Hi media
Mumbai Agarose was procured from Sisco Research Laboratories Pvt Ltd
Taq polymerase dNTPs and restriction enzymes were procured from Genei
Bangalore PCR purification kit was purchased from GE Healthcare UK
Ethidium bromide nitrocellulose membrane and nylon membrane were
procured from Sigma Aldrich Chemical Company MO US The designed
primers were synthesized by Sigma MO US Cloning kit was procured from
Fermentas Lifesciences EU DIG kit was obtained from Roche Company
Germany EDTA was procured from Qualigens Fine Chemicals All other
chemicals used in the study were procured from standard chemical
companies
43 Reagents and buffers
431 Reagents for plasmid isolation
Solution 1 50 mM Glucose 25 mM Tris- Cl (pH-80) 10 mM EDTA (pH-80)
Solution 2 02 N NaOH (freshly prepared from 10 N NaOH) 1 SDS
(Prepared freshly before use)
Chapter 2
235
Solution 3
50 M Potassium acetate 600mL
Glacial acetic acid 115mL
Distilled water 285mL
432 Ampicillin stock solution (100 mg mL)
Ampicillin was dissolved in distilled water filter sterilized and stored at
-20 0C
433 RNAse stock solution
10 mg of RNAse was dissolved in 1mL of distilled water and boiled for
15 min and used
434 CaCl2 solution
60 mM CaCl2 in MOPrsquos buffer (pH 65)
435 Luria Bertani broth
Tryptone peptone 100 g L
Yeast extract 50 g L
Sodium chloride 100 g L
The medium was autoclaved at 15 lbs 121 0C for 15 min
436 Tris- EDTA buffer (pH 80)
10 mM Tris Hcl (pH 80)
1 mM EDTA (pH 80)
pH was adjusted to 80 autoclaved and stored
437 Tris- acetate EDTA buffer (50X)
Tris buffer 242 g
05 M EDTA 10 mL
Glacial acetic acid 57 mL
Distilled water 84 mL
pH was adjusted to 80 autoclaved and stored
Chapter 2
236
438 DNA loading buffer
Bromophenol blue 025
Xylene Cyanol FF 025
Glycerol 30
This was prepared in double distilled water and stored in aliquots at -
20oC
439 Alkaline phosphatase buffer
Tris- HCl (pH 95) 100 mM
NaCl 100 mM
MgCl2 50 mM
1 M Tris (pH 95) 5 M NaCl and 1 M MgCl2 stocks were prepared in
double distilled water autoclaved and stored at RT Alkaline phosphatase
buffer was prepared by adding appropriate amounts of stock solutions
volume made up with double distilled water and stored at RT
4310 Maleic acid buffer
Maleic acid 100 mM
NaCl 150 mM
Maleic acid was dissolved in double distilled water containing NaCl
pH was adjusted to 7 with NaOH autoclaved and stored at RT
4311 Blocking solution
10 (wv) BSA was dissolved in maleic acid buffer by stirring and
heating autoclaved and stored at RT
4312 Colour development buffer
BCIP (50 mg mL) 70 μL
NBT (50 mg mL) 70 μL
Alkaline Phosphatase buffer 10 mL
50 mg mL NBT in 70 DMF and 50 mg mL BCIP in DMF were
prepared and stored at 4 0C protected from light BCIP may precipitate during
storage and should be warmed at RT to dissolve
Chapter 2
237
4313 Church hybridisation buffer
SDS 7 (wv)
BSA 1 (wv)
EDTA 1 mM
Na-PO4 (pH 74) 025 M
1M NaH2PO4 and 774mL of 1M Na2HPO4 were prepared and mixed to
produce 1M Na-PO4 pH 74 stock For long term storage this was
autoclaved and stored at 4 0C
The hybridization solution was prepared by dissolving 5 g BSA in
~100mL double distilled water 125 mL of 1M NaPO4 175 mL of 20 SDS
and 1 mL of 05 M EDTA were added The volume was made up to 500 mL
and stored at RT SDS precipitates out at cool temperature When this
happens the hybridization buffer was pre- warmed before use to redissolve
the SDS
4314 20X SSC
NaCl 3 M
Sodium citrate 03 M
1753 g of NaCl and 882 g of Sodium citrate were dissolved in ~700
mL of double distilled water The pH was adjusted to 70 with HCl and the
volume was made up to 1 L The solution was autoclaved and stored at RT
44 Methods
441 Microorganisms and culture conditions
All the members of the consortium were grown individually in Luria
Bertoni broth for 18 h under shaking conditions (180 rpm) Cells were
harvested by centrifuging at 10 000 rpm at 4 0C for 15 min The supernatant
was discarded and pellet was washed with minimal medium (331) induced
with 10 ppm DDT and used for the isolation of genomic and plasmid DNAs
442 Isolation of genomic DNA from the bacterial isolates
Genomic DNA was isolated using the method described below
Suspended a 30 h old induced culture (after centrifugation) in 500L
Chapter 2
238
lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated
RNase) and incubated at 37 0C for around 30 min or till the cells became
translucent To this was added 250 L of 2 SDS and vortexed gently to mix
until the viscosity of the solution decreased noticeably 250 L of neutral
chloroform solution was added to this and vortexed for 30 seconds spun for 2
min in micro centrifuge and the supernatant was removed leaving the white
interface behind This was repeated twice or till no or very little interface was
seen To this 01 volume of 3M sodium acetate (pH 48) was added and
mixed This was followed by the addition of 1 volume of isopropanol and
mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the
micro centrifuge Supernatant was poured off The pellet was redissolved in
500 L of TE buffer An agarose gel electrophoresis was carried out to check
the genomic DNA
443 Isolation of plasmid DNA from the bacterial isolates
Plasmid isolation was done according to Maniatis et al (1982)
Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria
Bertoni and harvested cells were induced with 10 ppm DDT The cells were
then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution
1 200 L of freshly prepared solution 2 was added and mixed 300 L of
solution 3 was added and centrifuged for 15 min The supernatant was
transferred to a fresh tube and equal volume of phenol- chloroform was
added and vortexed thoroughly The upper aqueous phase was transferred
to a fresh tube equal volume of chloroform was added and centrifuged at 10
000 rpm for 10 min to remove traces of phenol To the upper aqueous layer
2 volumes of absolute ethanol was added and kept at -20 0C for precipitation
The tube was centrifuged at 10000 rpm for 10 min supernatant was
discarded carefully The pellet was air- dried and dissolved in 20 L TE
buffer An agarose gel electrophoresis was carried out to check the plasmid
DNA To isolate cloned plasmids ampicillin was added during inoculation
The cells were not induced with DDT
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
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eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
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hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
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Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
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Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
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Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
234
which readily degraded DDT to DDD Wedemeyer (1967) studied DDT
metabolic pathway by incubation of proposed intermediates with organisms
and examining the products formed The metabolism of DDT in Aerobacter
aerogenes goes in order DDT DDD DDMU DDNU DDOH DDA
DBP and direct conversion of DDT to DDE (Wedemeyer 1967) Reports
on the involvement of enzymes in the degradation of DDT indicate the
presence of enzymes like dioxygenases (Nadeau et al 1994)
dehydrogenases (Bourquin 1977) oxygenases (Ahmed et al 1991) Only a
few enzymes have been described in DDT degradative pathway (Singh et al
1999) The conversion of DDT to DDD involves a dehydrochlorination step
In our laboratory an attempt was made to clone sequence and express the
gene responsible for this step
42 Materials
Luria- Bertoni broth and ampicillin were procured from Hi media
Mumbai Agarose was procured from Sisco Research Laboratories Pvt Ltd
Taq polymerase dNTPs and restriction enzymes were procured from Genei
Bangalore PCR purification kit was purchased from GE Healthcare UK
Ethidium bromide nitrocellulose membrane and nylon membrane were
procured from Sigma Aldrich Chemical Company MO US The designed
primers were synthesized by Sigma MO US Cloning kit was procured from
Fermentas Lifesciences EU DIG kit was obtained from Roche Company
Germany EDTA was procured from Qualigens Fine Chemicals All other
chemicals used in the study were procured from standard chemical
companies
43 Reagents and buffers
431 Reagents for plasmid isolation
Solution 1 50 mM Glucose 25 mM Tris- Cl (pH-80) 10 mM EDTA (pH-80)
Solution 2 02 N NaOH (freshly prepared from 10 N NaOH) 1 SDS
(Prepared freshly before use)
Chapter 2
235
Solution 3
50 M Potassium acetate 600mL
Glacial acetic acid 115mL
Distilled water 285mL
432 Ampicillin stock solution (100 mg mL)
Ampicillin was dissolved in distilled water filter sterilized and stored at
-20 0C
433 RNAse stock solution
10 mg of RNAse was dissolved in 1mL of distilled water and boiled for
15 min and used
434 CaCl2 solution
60 mM CaCl2 in MOPrsquos buffer (pH 65)
435 Luria Bertani broth
Tryptone peptone 100 g L
Yeast extract 50 g L
Sodium chloride 100 g L
The medium was autoclaved at 15 lbs 121 0C for 15 min
436 Tris- EDTA buffer (pH 80)
10 mM Tris Hcl (pH 80)
1 mM EDTA (pH 80)
pH was adjusted to 80 autoclaved and stored
437 Tris- acetate EDTA buffer (50X)
Tris buffer 242 g
05 M EDTA 10 mL
Glacial acetic acid 57 mL
Distilled water 84 mL
pH was adjusted to 80 autoclaved and stored
Chapter 2
236
438 DNA loading buffer
Bromophenol blue 025
Xylene Cyanol FF 025
Glycerol 30
This was prepared in double distilled water and stored in aliquots at -
20oC
439 Alkaline phosphatase buffer
Tris- HCl (pH 95) 100 mM
NaCl 100 mM
MgCl2 50 mM
1 M Tris (pH 95) 5 M NaCl and 1 M MgCl2 stocks were prepared in
double distilled water autoclaved and stored at RT Alkaline phosphatase
buffer was prepared by adding appropriate amounts of stock solutions
volume made up with double distilled water and stored at RT
4310 Maleic acid buffer
Maleic acid 100 mM
NaCl 150 mM
Maleic acid was dissolved in double distilled water containing NaCl
pH was adjusted to 7 with NaOH autoclaved and stored at RT
4311 Blocking solution
10 (wv) BSA was dissolved in maleic acid buffer by stirring and
heating autoclaved and stored at RT
4312 Colour development buffer
BCIP (50 mg mL) 70 μL
NBT (50 mg mL) 70 μL
Alkaline Phosphatase buffer 10 mL
50 mg mL NBT in 70 DMF and 50 mg mL BCIP in DMF were
prepared and stored at 4 0C protected from light BCIP may precipitate during
storage and should be warmed at RT to dissolve
Chapter 2
237
4313 Church hybridisation buffer
SDS 7 (wv)
BSA 1 (wv)
EDTA 1 mM
Na-PO4 (pH 74) 025 M
1M NaH2PO4 and 774mL of 1M Na2HPO4 were prepared and mixed to
produce 1M Na-PO4 pH 74 stock For long term storage this was
autoclaved and stored at 4 0C
The hybridization solution was prepared by dissolving 5 g BSA in
~100mL double distilled water 125 mL of 1M NaPO4 175 mL of 20 SDS
and 1 mL of 05 M EDTA were added The volume was made up to 500 mL
and stored at RT SDS precipitates out at cool temperature When this
happens the hybridization buffer was pre- warmed before use to redissolve
the SDS
4314 20X SSC
NaCl 3 M
Sodium citrate 03 M
1753 g of NaCl and 882 g of Sodium citrate were dissolved in ~700
mL of double distilled water The pH was adjusted to 70 with HCl and the
volume was made up to 1 L The solution was autoclaved and stored at RT
44 Methods
441 Microorganisms and culture conditions
All the members of the consortium were grown individually in Luria
Bertoni broth for 18 h under shaking conditions (180 rpm) Cells were
harvested by centrifuging at 10 000 rpm at 4 0C for 15 min The supernatant
was discarded and pellet was washed with minimal medium (331) induced
with 10 ppm DDT and used for the isolation of genomic and plasmid DNAs
442 Isolation of genomic DNA from the bacterial isolates
Genomic DNA was isolated using the method described below
Suspended a 30 h old induced culture (after centrifugation) in 500L
Chapter 2
238
lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated
RNase) and incubated at 37 0C for around 30 min or till the cells became
translucent To this was added 250 L of 2 SDS and vortexed gently to mix
until the viscosity of the solution decreased noticeably 250 L of neutral
chloroform solution was added to this and vortexed for 30 seconds spun for 2
min in micro centrifuge and the supernatant was removed leaving the white
interface behind This was repeated twice or till no or very little interface was
seen To this 01 volume of 3M sodium acetate (pH 48) was added and
mixed This was followed by the addition of 1 volume of isopropanol and
mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the
micro centrifuge Supernatant was poured off The pellet was redissolved in
500 L of TE buffer An agarose gel electrophoresis was carried out to check
the genomic DNA
443 Isolation of plasmid DNA from the bacterial isolates
Plasmid isolation was done according to Maniatis et al (1982)
Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria
Bertoni and harvested cells were induced with 10 ppm DDT The cells were
then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution
1 200 L of freshly prepared solution 2 was added and mixed 300 L of
solution 3 was added and centrifuged for 15 min The supernatant was
transferred to a fresh tube and equal volume of phenol- chloroform was
added and vortexed thoroughly The upper aqueous phase was transferred
to a fresh tube equal volume of chloroform was added and centrifuged at 10
000 rpm for 10 min to remove traces of phenol To the upper aqueous layer
2 volumes of absolute ethanol was added and kept at -20 0C for precipitation
The tube was centrifuged at 10000 rpm for 10 min supernatant was
discarded carefully The pellet was air- dried and dissolved in 20 L TE
buffer An agarose gel electrophoresis was carried out to check the plasmid
DNA To isolate cloned plasmids ampicillin was added during inoculation
The cells were not induced with DDT
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
235
Solution 3
50 M Potassium acetate 600mL
Glacial acetic acid 115mL
Distilled water 285mL
432 Ampicillin stock solution (100 mg mL)
Ampicillin was dissolved in distilled water filter sterilized and stored at
-20 0C
433 RNAse stock solution
10 mg of RNAse was dissolved in 1mL of distilled water and boiled for
15 min and used
434 CaCl2 solution
60 mM CaCl2 in MOPrsquos buffer (pH 65)
435 Luria Bertani broth
Tryptone peptone 100 g L
Yeast extract 50 g L
Sodium chloride 100 g L
The medium was autoclaved at 15 lbs 121 0C for 15 min
436 Tris- EDTA buffer (pH 80)
10 mM Tris Hcl (pH 80)
1 mM EDTA (pH 80)
pH was adjusted to 80 autoclaved and stored
437 Tris- acetate EDTA buffer (50X)
Tris buffer 242 g
05 M EDTA 10 mL
Glacial acetic acid 57 mL
Distilled water 84 mL
pH was adjusted to 80 autoclaved and stored
Chapter 2
236
438 DNA loading buffer
Bromophenol blue 025
Xylene Cyanol FF 025
Glycerol 30
This was prepared in double distilled water and stored in aliquots at -
20oC
439 Alkaline phosphatase buffer
Tris- HCl (pH 95) 100 mM
NaCl 100 mM
MgCl2 50 mM
1 M Tris (pH 95) 5 M NaCl and 1 M MgCl2 stocks were prepared in
double distilled water autoclaved and stored at RT Alkaline phosphatase
buffer was prepared by adding appropriate amounts of stock solutions
volume made up with double distilled water and stored at RT
4310 Maleic acid buffer
Maleic acid 100 mM
NaCl 150 mM
Maleic acid was dissolved in double distilled water containing NaCl
pH was adjusted to 7 with NaOH autoclaved and stored at RT
4311 Blocking solution
10 (wv) BSA was dissolved in maleic acid buffer by stirring and
heating autoclaved and stored at RT
4312 Colour development buffer
BCIP (50 mg mL) 70 μL
NBT (50 mg mL) 70 μL
Alkaline Phosphatase buffer 10 mL
50 mg mL NBT in 70 DMF and 50 mg mL BCIP in DMF were
prepared and stored at 4 0C protected from light BCIP may precipitate during
storage and should be warmed at RT to dissolve
Chapter 2
237
4313 Church hybridisation buffer
SDS 7 (wv)
BSA 1 (wv)
EDTA 1 mM
Na-PO4 (pH 74) 025 M
1M NaH2PO4 and 774mL of 1M Na2HPO4 were prepared and mixed to
produce 1M Na-PO4 pH 74 stock For long term storage this was
autoclaved and stored at 4 0C
The hybridization solution was prepared by dissolving 5 g BSA in
~100mL double distilled water 125 mL of 1M NaPO4 175 mL of 20 SDS
and 1 mL of 05 M EDTA were added The volume was made up to 500 mL
and stored at RT SDS precipitates out at cool temperature When this
happens the hybridization buffer was pre- warmed before use to redissolve
the SDS
4314 20X SSC
NaCl 3 M
Sodium citrate 03 M
1753 g of NaCl and 882 g of Sodium citrate were dissolved in ~700
mL of double distilled water The pH was adjusted to 70 with HCl and the
volume was made up to 1 L The solution was autoclaved and stored at RT
44 Methods
441 Microorganisms and culture conditions
All the members of the consortium were grown individually in Luria
Bertoni broth for 18 h under shaking conditions (180 rpm) Cells were
harvested by centrifuging at 10 000 rpm at 4 0C for 15 min The supernatant
was discarded and pellet was washed with minimal medium (331) induced
with 10 ppm DDT and used for the isolation of genomic and plasmid DNAs
442 Isolation of genomic DNA from the bacterial isolates
Genomic DNA was isolated using the method described below
Suspended a 30 h old induced culture (after centrifugation) in 500L
Chapter 2
238
lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated
RNase) and incubated at 37 0C for around 30 min or till the cells became
translucent To this was added 250 L of 2 SDS and vortexed gently to mix
until the viscosity of the solution decreased noticeably 250 L of neutral
chloroform solution was added to this and vortexed for 30 seconds spun for 2
min in micro centrifuge and the supernatant was removed leaving the white
interface behind This was repeated twice or till no or very little interface was
seen To this 01 volume of 3M sodium acetate (pH 48) was added and
mixed This was followed by the addition of 1 volume of isopropanol and
mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the
micro centrifuge Supernatant was poured off The pellet was redissolved in
500 L of TE buffer An agarose gel electrophoresis was carried out to check
the genomic DNA
443 Isolation of plasmid DNA from the bacterial isolates
Plasmid isolation was done according to Maniatis et al (1982)
Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria
Bertoni and harvested cells were induced with 10 ppm DDT The cells were
then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution
1 200 L of freshly prepared solution 2 was added and mixed 300 L of
solution 3 was added and centrifuged for 15 min The supernatant was
transferred to a fresh tube and equal volume of phenol- chloroform was
added and vortexed thoroughly The upper aqueous phase was transferred
to a fresh tube equal volume of chloroform was added and centrifuged at 10
000 rpm for 10 min to remove traces of phenol To the upper aqueous layer
2 volumes of absolute ethanol was added and kept at -20 0C for precipitation
The tube was centrifuged at 10000 rpm for 10 min supernatant was
discarded carefully The pellet was air- dried and dissolved in 20 L TE
buffer An agarose gel electrophoresis was carried out to check the plasmid
DNA To isolate cloned plasmids ampicillin was added during inoculation
The cells were not induced with DDT
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
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Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
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2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
236
438 DNA loading buffer
Bromophenol blue 025
Xylene Cyanol FF 025
Glycerol 30
This was prepared in double distilled water and stored in aliquots at -
20oC
439 Alkaline phosphatase buffer
Tris- HCl (pH 95) 100 mM
NaCl 100 mM
MgCl2 50 mM
1 M Tris (pH 95) 5 M NaCl and 1 M MgCl2 stocks were prepared in
double distilled water autoclaved and stored at RT Alkaline phosphatase
buffer was prepared by adding appropriate amounts of stock solutions
volume made up with double distilled water and stored at RT
4310 Maleic acid buffer
Maleic acid 100 mM
NaCl 150 mM
Maleic acid was dissolved in double distilled water containing NaCl
pH was adjusted to 7 with NaOH autoclaved and stored at RT
4311 Blocking solution
10 (wv) BSA was dissolved in maleic acid buffer by stirring and
heating autoclaved and stored at RT
4312 Colour development buffer
BCIP (50 mg mL) 70 μL
NBT (50 mg mL) 70 μL
Alkaline Phosphatase buffer 10 mL
50 mg mL NBT in 70 DMF and 50 mg mL BCIP in DMF were
prepared and stored at 4 0C protected from light BCIP may precipitate during
storage and should be warmed at RT to dissolve
Chapter 2
237
4313 Church hybridisation buffer
SDS 7 (wv)
BSA 1 (wv)
EDTA 1 mM
Na-PO4 (pH 74) 025 M
1M NaH2PO4 and 774mL of 1M Na2HPO4 were prepared and mixed to
produce 1M Na-PO4 pH 74 stock For long term storage this was
autoclaved and stored at 4 0C
The hybridization solution was prepared by dissolving 5 g BSA in
~100mL double distilled water 125 mL of 1M NaPO4 175 mL of 20 SDS
and 1 mL of 05 M EDTA were added The volume was made up to 500 mL
and stored at RT SDS precipitates out at cool temperature When this
happens the hybridization buffer was pre- warmed before use to redissolve
the SDS
4314 20X SSC
NaCl 3 M
Sodium citrate 03 M
1753 g of NaCl and 882 g of Sodium citrate were dissolved in ~700
mL of double distilled water The pH was adjusted to 70 with HCl and the
volume was made up to 1 L The solution was autoclaved and stored at RT
44 Methods
441 Microorganisms and culture conditions
All the members of the consortium were grown individually in Luria
Bertoni broth for 18 h under shaking conditions (180 rpm) Cells were
harvested by centrifuging at 10 000 rpm at 4 0C for 15 min The supernatant
was discarded and pellet was washed with minimal medium (331) induced
with 10 ppm DDT and used for the isolation of genomic and plasmid DNAs
442 Isolation of genomic DNA from the bacterial isolates
Genomic DNA was isolated using the method described below
Suspended a 30 h old induced culture (after centrifugation) in 500L
Chapter 2
238
lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated
RNase) and incubated at 37 0C for around 30 min or till the cells became
translucent To this was added 250 L of 2 SDS and vortexed gently to mix
until the viscosity of the solution decreased noticeably 250 L of neutral
chloroform solution was added to this and vortexed for 30 seconds spun for 2
min in micro centrifuge and the supernatant was removed leaving the white
interface behind This was repeated twice or till no or very little interface was
seen To this 01 volume of 3M sodium acetate (pH 48) was added and
mixed This was followed by the addition of 1 volume of isopropanol and
mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the
micro centrifuge Supernatant was poured off The pellet was redissolved in
500 L of TE buffer An agarose gel electrophoresis was carried out to check
the genomic DNA
443 Isolation of plasmid DNA from the bacterial isolates
Plasmid isolation was done according to Maniatis et al (1982)
Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria
Bertoni and harvested cells were induced with 10 ppm DDT The cells were
then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution
1 200 L of freshly prepared solution 2 was added and mixed 300 L of
solution 3 was added and centrifuged for 15 min The supernatant was
transferred to a fresh tube and equal volume of phenol- chloroform was
added and vortexed thoroughly The upper aqueous phase was transferred
to a fresh tube equal volume of chloroform was added and centrifuged at 10
000 rpm for 10 min to remove traces of phenol To the upper aqueous layer
2 volumes of absolute ethanol was added and kept at -20 0C for precipitation
The tube was centrifuged at 10000 rpm for 10 min supernatant was
discarded carefully The pellet was air- dried and dissolved in 20 L TE
buffer An agarose gel electrophoresis was carried out to check the plasmid
DNA To isolate cloned plasmids ampicillin was added during inoculation
The cells were not induced with DDT
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Bourquin AW 1977 Degradation of malathion by salt- marsh
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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
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2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
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Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
237
4313 Church hybridisation buffer
SDS 7 (wv)
BSA 1 (wv)
EDTA 1 mM
Na-PO4 (pH 74) 025 M
1M NaH2PO4 and 774mL of 1M Na2HPO4 were prepared and mixed to
produce 1M Na-PO4 pH 74 stock For long term storage this was
autoclaved and stored at 4 0C
The hybridization solution was prepared by dissolving 5 g BSA in
~100mL double distilled water 125 mL of 1M NaPO4 175 mL of 20 SDS
and 1 mL of 05 M EDTA were added The volume was made up to 500 mL
and stored at RT SDS precipitates out at cool temperature When this
happens the hybridization buffer was pre- warmed before use to redissolve
the SDS
4314 20X SSC
NaCl 3 M
Sodium citrate 03 M
1753 g of NaCl and 882 g of Sodium citrate were dissolved in ~700
mL of double distilled water The pH was adjusted to 70 with HCl and the
volume was made up to 1 L The solution was autoclaved and stored at RT
44 Methods
441 Microorganisms and culture conditions
All the members of the consortium were grown individually in Luria
Bertoni broth for 18 h under shaking conditions (180 rpm) Cells were
harvested by centrifuging at 10 000 rpm at 4 0C for 15 min The supernatant
was discarded and pellet was washed with minimal medium (331) induced
with 10 ppm DDT and used for the isolation of genomic and plasmid DNAs
442 Isolation of genomic DNA from the bacterial isolates
Genomic DNA was isolated using the method described below
Suspended a 30 h old induced culture (after centrifugation) in 500L
Chapter 2
238
lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated
RNase) and incubated at 37 0C for around 30 min or till the cells became
translucent To this was added 250 L of 2 SDS and vortexed gently to mix
until the viscosity of the solution decreased noticeably 250 L of neutral
chloroform solution was added to this and vortexed for 30 seconds spun for 2
min in micro centrifuge and the supernatant was removed leaving the white
interface behind This was repeated twice or till no or very little interface was
seen To this 01 volume of 3M sodium acetate (pH 48) was added and
mixed This was followed by the addition of 1 volume of isopropanol and
mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the
micro centrifuge Supernatant was poured off The pellet was redissolved in
500 L of TE buffer An agarose gel electrophoresis was carried out to check
the genomic DNA
443 Isolation of plasmid DNA from the bacterial isolates
Plasmid isolation was done according to Maniatis et al (1982)
Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria
Bertoni and harvested cells were induced with 10 ppm DDT The cells were
then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution
1 200 L of freshly prepared solution 2 was added and mixed 300 L of
solution 3 was added and centrifuged for 15 min The supernatant was
transferred to a fresh tube and equal volume of phenol- chloroform was
added and vortexed thoroughly The upper aqueous phase was transferred
to a fresh tube equal volume of chloroform was added and centrifuged at 10
000 rpm for 10 min to remove traces of phenol To the upper aqueous layer
2 volumes of absolute ethanol was added and kept at -20 0C for precipitation
The tube was centrifuged at 10000 rpm for 10 min supernatant was
discarded carefully The pellet was air- dried and dissolved in 20 L TE
buffer An agarose gel electrophoresis was carried out to check the plasmid
DNA To isolate cloned plasmids ampicillin was added during inoculation
The cells were not induced with DDT
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
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structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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Junker F Cook AM 1997 Conjugative plasmids and the degradation of
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Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
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pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
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intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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element IS1247 Journal of Bacteriology 177 1348- 1356
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
238
lysozyme solution (containing 2mgmL lysozyme and 50mgmL heat treated
RNase) and incubated at 37 0C for around 30 min or till the cells became
translucent To this was added 250 L of 2 SDS and vortexed gently to mix
until the viscosity of the solution decreased noticeably 250 L of neutral
chloroform solution was added to this and vortexed for 30 seconds spun for 2
min in micro centrifuge and the supernatant was removed leaving the white
interface behind This was repeated twice or till no or very little interface was
seen To this 01 volume of 3M sodium acetate (pH 48) was added and
mixed This was followed by the addition of 1 volume of isopropanol and
mixing Contents were incubated for 24 h at -20 0C and spun for 5 min in the
micro centrifuge Supernatant was poured off The pellet was redissolved in
500 L of TE buffer An agarose gel electrophoresis was carried out to check
the genomic DNA
443 Isolation of plasmid DNA from the bacterial isolates
Plasmid isolation was done according to Maniatis et al (1982)
Single colonies of appropriate strain grown for 24 h inoculated to 2 mL Luria
Bertoni and harvested cells were induced with 10 ppm DDT The cells were
then harvested by centrifuging at 8000 rpm for 5 min and lysed with solution
1 200 L of freshly prepared solution 2 was added and mixed 300 L of
solution 3 was added and centrifuged for 15 min The supernatant was
transferred to a fresh tube and equal volume of phenol- chloroform was
added and vortexed thoroughly The upper aqueous phase was transferred
to a fresh tube equal volume of chloroform was added and centrifuged at 10
000 rpm for 10 min to remove traces of phenol To the upper aqueous layer
2 volumes of absolute ethanol was added and kept at -20 0C for precipitation
The tube was centrifuged at 10000 rpm for 10 min supernatant was
discarded carefully The pellet was air- dried and dissolved in 20 L TE
buffer An agarose gel electrophoresis was carried out to check the plasmid
DNA To isolate cloned plasmids ampicillin was added during inoculation
The cells were not induced with DDT
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
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Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
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Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
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Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
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van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
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van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
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Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
239
444 Designing of the oligonucleotide primers
DDT dehydrochlorinase sequences available in the databank were
aligned using Dialign 20 software program and primers were designed using
Primer 30 program and the primer sequence is given in Table 41
Table 41 Nucleotide sequences of the primers used for
screening DDT- dhl1 producing bacterial spp
Primer Primer name Primer sequence
Forward primer dhl1F CTCGAGGCGATTGGCCGCGGCATAAC
Reverse primer dhl1R AAGCTTAAATTTCGGTTTCGGCACGCT
445 PCR amplification of DDT- dhl1 gene from Pseudomonas putida
The PCR amplification was carried out with genomic DNA and plasmid
DNA with Takara thermocycler unit The PCR mixture consisted of
Sterile deionised water 1898 L
Taq Buffer 25 L
dNTP mix 05 L
Taq Polymerase 03 L
Forward Primer 10 L
Reverse Primer 10 L
Template DNA plasmid DNA 10 L
PCR conditions used are as follows
4451 Gradient PCR
A gradient PCR was done with different temperatures from 50- 70 0C
720C 720C940C 940C
500 045
045
50- 700C
100 800
40C
25 Cycles
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
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Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
240
4452 Touchdown PCR
A touchdown PCR was done as programmed below
4453 PCR with optimised conditions
The amplified PCR products were analysed by agarose gel
electrophoresis
446 Analysis of the PCR products
The gel casting tray was set up with the appropriate comb Agarose
(1) was dissolved in 1X TAE buffer by heating It was cooled to 50 to 60 0C
and was poured into the gel casting unit The comb was removed carefully
from the solidified gel The agarose gel was taken in the electrophoresis tank
containing 1X electrophoresis buffer The wells were loaded with the DNA
samples mixed with gel loading buffer Markers (100 bp markers) were run
along with samples unless otherwise stated After 3- 4 h run at 40 V the gels
were stained with 1 ethidium bromide solution for 10 min and then
destained in double distilled water The DNA was visualised as bands under
U V transilluminator and documented
720C 72 0C 72 0C 4 0C
infin
045
100 030
940C 940C 600C 940C 500C
500 030
10 cycles 30 cycles
045
100 15
72 0C 94 0 C 94 0 C
500 045
115
55 0C
100 800
4 0 C
30 Cycles
72 0C
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
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Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
241
447 Purification of amplicons of DDT- dhl 1 gene of Pseudomonas
putida T5
The obtained PCR product was purified by using GE gel purification kit
The bands were excised from the agarose gel by cutting with a clean blade
under UV light and transferred into a 20 mL microcentrifuge tube The
following steps were involved in purification of the DNA bands 10 microL capture
buffer type 3 was added for each 10 mg of agarose gel slice The contents of
the tubes were mixed by inverting the tubes and kept in water bath at 60 0C
till agarose completely dissolved GFXtm microspin column was placed in 20
mL collection tube provided by the kit For binding the DNA the sample was
passed though the column and centrifuged at 8 000 rpm for 60 sec The flow
through liquid was discarded and the GFXtm microspin column was placed in
new 20mL collection tube The same procedure was repeated until the entire
sample was poured Now 500 microL of wash buffer type1was added and
centrifuged at 8 000 rpm for 30 sec The collection tube was discarded and
GFXtm microspin column was transferred to clean 15 mL DNase free
microcentrifuge tube 10- 50microL of elution buffer type 6 (nuclease free water)
was added and kept at RT for 60 sec Then it was centrifuged at 8 000 rpm
for 60 sec to elute the DNA The obtained purified DNA sample was stored at
-200C and checked by agarose gel electrophoresis
448 Cloning of DDT- dhl1 gene of Pseudomonas putida T5
4481 Ligation of the purified PCR amplicon into pTZ57RT vector
Ligation of the purified PCR amplicon was done by using Fermentas
kit To a 20 mL microcentrifuge tube 3 microL vector pTZ57RT (018ρ mol
ends) (Fig41) 6 microL 5X ligation buffer 4 microL PCR product (054ρ mol ends)
16 microL nuclease free water and 1 microL T4 DNA ligase were added and incubated
at 4 0C overnight
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
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Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
242
Fig41 Vector map of pTZ57RT
4482 Transformation of E coli DH5α
44821 Preparation of competent cells
A single colony of E coli DH5α was inoculated to 50 mL of Luria
Bertoni broth and incubated overnight at 37 0C in a shaker The cells were
transferred aseptically to sterile tubes and harvested at 10 000 rpm 10 min
4 0C The above cells were then treated with 10 mL of 60 mM CaCl2 in MOPrsquos
buffer at a pH 65 and vortexed for few minutes The tubes were centrifuged
at 10 000 rpm 4 0C for 10 min The supernatant was discarded and the
pellet was resuspended in the 4 mL solution containing 01 M CaCl2 The
tubes were kept at 4 0C overnight
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
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eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
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hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
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Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
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Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
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Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
243
44822 Transformation of competent cells
Transformation of competent cells into E coli DH5α was done by using
Fermentas transformation kit 150 microL of overnight stored competent cells
were taken and 15 mL of C- media was added The tubes were incubated at
37 0C for 20 min The tubes were centrifuged at 8 000 rpm 4 0C for 1 min
and supernatant was discarded The pellet was suspended in 300 μL of T-
solution The tubes were placed on ice for 5 min Again the tubes were
centrifuged at 8 000 rpm 4 0C for 1 min and supernatant was discarded
The pellet was resuspended in 120 μL of T- solution and kept in ice for 5 min
To 50 μL of above solution 25 μL of ligation mixture containing 14 ng vector
DNA was added and kept in ice for 5 min The solution was poured to pre-
warmed Luria Bertoni media and incubated at 37 0C overnight
449 Selection of transformants
100 μL of transformation mix was plated onto Luria Bertoni agar plates
containing 100 μL mL ampicillin The plates were incubated at 37 0C
overnight for the colonies to grow
4410 Analysis of the transformants
The plasmid profile of the transformed colonies was checked by gel
electrophoresis of plasmids with and without insert
4411 Isolation of plasmid DNA from transformed colonies
The transformed cells in Luria Bertoni broth were taken in 2 mL micro
centrifuge tube and plasmid isolation was done as described under section
443 An agarose gel electrophoresis was carried out to check the
transformation
4412 Sequencing of the cloned genestransformed plasmids
The transformed plasmid pTZ57RT containing the DDT-dhl 1 insert
was sent for sequencing to Bio-serve Biotechnologies Pvt Ltd Hyderabad
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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Junker F Cook AM 1997 Conjugative plasmids and the degradation of
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
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intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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element IS1247 Journal of Bacteriology 177 1348- 1356
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
244
4413 Sub- cloning of DDT- dhl1 gene of Pseudomonas putida T5
44131 Isolation of plasmid DNA from transformed colonies
Plasmid DNA from the transformed colonies was isolated by alkali lysis
as described under section 4411
44132 Restriction digestion of recombinant plasmids for insert release
The cloned vector was double digested with EcoR1 and Hind III for the
release of cloned insert The restriction digestion was carried out as per
suppliersrsquo instructions After the addition of restriction enzyme along with its
buffer the tubes were incubated at 37 0C for 1 h The insert release was
cross- checked on agarose gel electrophoresis
44133 Sub- cloning the DDT- dhl1 insert into E coli DH5α
The insert DNA was ligated to pUC18 (Fig42) vector and also pET28a
as per the procedure described under 4413 The cloned plasmid pUC 18
was transformed into E coli DH5α and pET28a (Fig43) to E coli BL21
according to 44822 The transformants were selected on Luria Bertoni
agar ampicillin plates The E coli BL21 transformed with pET28a vector were
selected with IPTG- X- Gal selection
Fig42 Vector map of pUC18
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
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Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Bourquin AW 1977 Degradation of malathion by salt- marsh
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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
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reductase fold without an NAD (P) H binding site European Molecular
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
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evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
245
Fig43 Vector map of pET28a
44134 Isolation of DDT- dhl1 enzyme
The cells of E coli were grown on Luria Bertoni broth containing
ampicillin for 6 h and then were induced with IPTG (04 mM) The cells were
harvested after 16 h by centrifugation at 10 000 rpm 4 0C for 10 min Cells
were washed with phosphate buffer The crude extracts of recombinant E
coli were prepared by sonication The supernatant was purified by using
HIS- tag purification kit The eluent was subjected to electrophoresis on a
12 (wv) SDS- PAGE The amount of protein was determined by
determining O D at 280 nm The enzyme was estimated for activity in micro
titre plate The assay was based on decrease in the pH of a weakly buffered
medium containing phenol red as an indicator dye The colour change from
reddish orange to yellow which occur when Cl- is released because of
enzyme action on DDT was identified as the enzyme activity The culture
showing dehydrohalogenase activity in micro titre plate was tested for DDT
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
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2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Bourquin AW 1977 Degradation of malathion by salt- marsh
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362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
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2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
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41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
246
clearance assay on minimal- DDT- agar plate to conform the activity Spray
plates were prepared with 15 agar in minimal medium on Petri dishes
The cloned isolate was streaked on to the plate The surface of the plate was
sprayed with 01 DDT as acetone solution The plates were incubated at
37 0C for 4- 5 days The formation of clearance surrounding the colonies was
used as an indicator of DDT-dehydrohalogenase activity
4414 Labelling DDT- dhl1 gene by using DIG kit (digoxigenin- dUTP
alkali labile labelling)
The labelling of the DDT- dhl1 gene was done by using DIG kit The
components of the kit are given in Table 42 For the labelling of the DDT-
dhl1 gene of Pseudomonas putida T5 PCR was carried out as per the
procedure 4453 and the obtained PCR product was purified by the
procedure given in 447
The O D of the purified product was determined by using
spectrophotometer (Schimadzu Japan) at 260 280 nm
10 ng- 3 μg of DNA was taken and sterile double distilled water was
added and the final volume was made up to 15 μL The DNA was denatured
by heating in a boiling bath for 10 min and quickly chilled in ice for 10 min
To this 2 μL of hexanucleotide mix (vial 5) 2 μL of dNTP labelling mix (vial 6)
and 1μl of Klenow enzyme (vial 7) were added mixed and centrifuged briefly
The reaction vials were incubated at 37 0C for 20 h After 20 h the reaction
was stopped by adding 2 μL of 02 M EDTA (pH 80) The labelled probe was
stored at -20 0C
4415 Restriction digestion of genomic DNA
For the restriction digestion of genomic DNA of Pseudomonas putida
T5 the restriction enzymes used are given in Table 43
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
247
Table 42 Components of DIG kit
Bottlecap
Label Content (including function)
1 Unlabelled
control DNA
20 μL unlabelled control DNA1 [100 μg mL]
10 mM Tris- HCl1 mM EDTA pH 80
Mixture of pBR328 DNA digested separately with
Bam H1 Bgl 1and Hinf 1 The separate digests
are combined in a ratio of 2 3 3
Sizes of 16 pBR328 fragments 490 2176 1766
1230 1033 653 394 298(2X) 234 (2X) 220 and
154 (2X) bp
Clear solution
Controls target in a southern blot
2 Unlabelled
controlled DNA
20 μL unlabelled control DNA2
[200 μg mL]
pBR328 DNA that has been linearized with Bam H1
Clear solution
To practice labelling and to check labelling
efficiency
3 DNA dilution
buffer
2 x 1mL DNA dilution buffer
[50 μg mL herring sperm DNA in 10 mM Tris-HCl
1 mM EDTA pH 80 (20 0C)]
Clear solution
For the dilution steps in the semi- quantitative
determination of labelling efficiency
4 Labelled control
DNA
50μl labelled control DNA
Linearized pBR328 DNA labelled with digoxigenin
according to the standard protocol
1μg template DNA and aprox 250ng digoxigenin-
labelled DNA
Clear solution
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
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Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
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Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
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van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
248
Table 43 Restriction enzymes used for digestion
Sl No Type of restriction enzyme
1 Sau 3A
2 Bgl ΙΙ
3 Sal І
4 Bam H І
5 Xho І
6 Hind Ш
7 Pvu ΙΙ
Estimation of the yield of DIG- labelled DNA
5 Hexa-
nucleotide mix
80 μL of 10X conc Hexanucleotide mix
[625 A260 unitsmL] random hexanucleotides
500 mM Tris-HCl 100 mM MgCl2 1 mM
Dithioerythritol [DTE] 2 mg mL BSA pH72
Clear solution
Component of the labelling reaction
6 dNTP Labelling
Mix
80 μL of 10X conc dNTP Labelling mix
1 mM dATP 1 mM dCTP 1 mM dGTP 065 mM
dTTP 035 mM DIG- 11- dUTP alkaline- labile
pH75 ( 20 0C)]
Clear solution
Component of the labelling reaction
7 Klenow Enzyme
Labelling Grade
40 μL Klenow Enzyme Labelling Grade
[2 units μL DNA Polymerase 1 (Klenow Enzyme
large fragment)]
Clear solution
Synthesis of DIG- labelled DNA
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Bourquin AW 1977 Degradation of malathion by salt- marsh
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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
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2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
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Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
249
To 6 μL of genomic DNA taken in separate vials 2 μL each of enzyme
buffer and enzyme were added respectively The vials were kept in the water
bath at 37 0C for 24 h
Agarose gel electrophoresis was carried out for the restriction digested
genomic DNA of Pseudomonas putida T5 For the restriction digested
genomic DNA southern blot analysis was carried out
4416 Southern blot analysis of DDT- dhl1 gene
The conventional transfer method introduced by Southern relies on a
gel-sandwich setup The southern blot was carried out as follows the gel
was placed over a buffer-soaked wick and overlaid with a piece of transfer
membrane A stack of dry paper towels was then placed on the top of the gel
sandwich to create a capillary action that ldquopullsrdquo the buffer from the wick
through the gel and the membrane and up towards the dry paper towels
Fractioned DNA fragments in the gel will be carried upward by capillary buffer
flow and retained on the membrane thus generating an imprint that is
identical to the electrophoresis pattern of the gel After 18 h of transfer the
DNA was then permanently fixed on the membrane by using U V cross-
linking Capillary action was usually conducted with a neutral high salt 20X
SSC buffer The membrane was removed and kept in the hybridization bottle
containing 10 mL hybridization buffer (4313) for 1 h at 65 0C 10 μL of the
probe was taken in a fresh Eppendorf tube and placed in boiling water for 10
min and immediately chilled on ice The denatured probe was added to 15
mL fresh hybridization solution and the hybridization was carried out overnight
at 65 0C The hybridization solution was discarded and the membrane was
thoroughly rinsed with 10 mL of 2X SSC washing buffer The washed
membrane was transferred into a clean dish containing maleic acid buffer and
the membrane was washed for 2- 5 min with gentle shaking at RT The
membrane was then replaced in the freshly prepared blocking solution and
incubated with shaking for 60 min The blocking solution was discarded and
diluted anti-DIG- AP antibody was added and the membrane was incubated
at RT for 40- 60 min with gentle shaking on the rocker The membrane was
then washed with maleic acid buffer for 20 min with shaking and this step
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
250
was repeated once by using the fresh washing tray The membrane was
equilibrated in the Alkaline Phosphatase buffer with gentle shacking The
colour development solution was added to the membrane and incubated in
the dark at RT for 30 min The colour development was monitored with clear
zones
4417 Production and isolation of antibody
Twenty week old Single comb white leg horn poultry (layers) were
immunized with purified DDT- dehydrochlorinase emulsified with Freundrsquos
complete adjuvant 250 g of DDT- dehydrochlorinase in PBS buffer (05 mL)
was mixed well with 05 mL of Freundrsquos complete adjuvant and injected to
poultry intramuscularly (at breast muscle) Subsequent immunizations were
done with Freundrsquos incomplete adjuvant and 500 microg of enzyme protein every
15 days for 6 months
Egg yolk antibody (IgY) was isolated by PEG method Yolk was
separated from egg albumin 40 mL of phosphate buffer saline (50 mM pH
72) was added for per yolk and stirred for 1 h at RT 10 mL of chloroform
yolk was added and stirring was continued for another 30 min at RT The
precipitate formed was removed by centrifugation To the supernatant 14
(wv) polyethylene glycol 6000 was added and stirred for 90 min at RT The
solution was centrifuged at 4 0C 10 000 rpm for 10 min The precipitate thus
obtained (which contains IgY) was dissolved in a known quantity of
phosphate buffer saline (50 mM pH 72) and dialyzed against distilled water
for 24h at 4 0C to obtain purified antibodies
4418 Western blot analysis of DDT- dhl1 gene
44181 SDS- PAGE was done according to Laemmli (1970)
44182 Electrophoretic transfer of SDS-PAGE gel to nitrocellulose
membrane
Whatman No3 filter papers were cut according to the gel size The
nitrocellulose membrane was cut to the same size of SDS-PAGE gel The
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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cluster encoding haloalkane catabolism Journal of Bacteriology 182
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
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intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
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Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
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Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
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van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
251
filter papers containing 8 sheets were wetted in transfer solution Glycine 15
Tris 29 SDS 0187 methanol 100 mL and water to 1L) and placed on the
transfer unit Then nitrocellulose membrane was placed on the filter papers
and then gel was placed over it 8 sheets of Whatman No 3 were placed over
the gel The current passed through at the rate of 08mA cm2 for 75 min A
part of membrane was stained with amido black (002 in distilled water) and
rest was taken for western blot
44183 Western blot
SDS-PAGE transferred Nitrocellulose membrane was blocked with 2
gelatin for 2 hrs and washed with TBST buffer for 10 min with three changes
The membrane was incubated with the primary antibody (IgY 15000 in
TBST) with shaking at RT for 2 hrs Then the membrane was washed for 10
min with three changes in TBST buffer Then the membrane was incubated
with the secondary antibody Goat anti chicken IgY conjugated to alkaline
phosphatase (15000 in TBST) with shaking at RT for 1 hr followed by
washing with TBST for 10 min with three changes
44184 Colour development
To 10 mL of alkaline phosphatase buffer pH 95 (100 mM NaCl 5 mM
MgCl2 100 mM Tris) 66 L of NBT (25mg dissolved in 500 L of 70
dimethyl formamide) and 33 L of BCIP (25mg dissolved in 100 dimethyl
formamide) were added as substrates The colour development reaction was
stopped by adding 200 L of 05 M EDTA pH 80 in 50 mL 09 NaCl or
simply by replacing the buffer by water
45 Results
451 Screening for DDT- dhl 1 producers
Genomic and plasmid DNA from all the ten bacterial isolates were
screened by gradient PCR and touch- down PCR for the presence of DDT-
dhl 1 gene by PCR using DDT- dehydrohalogenase primer pairs described in
Table 41 All the isolates gave positive amplification with genomic DNA
(Fig44) and plasmid DNA (Fig45) The samples were run on 1 agarose
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
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Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
252
gel Distinct PCR products of around 750- 800 bp were obtained In our
studies genomic DNA of Pseudomonas putida T5 was chosen for further
work
Fig 44 Agarose gel electrophoresis of PCR products of genomic DNA
Lane 1-10 PCR products of genomic DNA of bacterial isolates T1 to T10
Fig45 Agarose gel electrophoresis of PCR products of plasmid DNA
Lane 1- 10 PCR products of plasmid DNA of bacterial isolates T1 to T10
To optimise the PCR conditions gradient PCR between 50- 70 0C was
carried out But in all the temperatures the amplification was not much
(Fig46) The touch- down PCR conditions also did not give better
amplification (Fig46) The PCR done with optimised conditions of
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
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intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
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Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
253
denaturation temperature 94 0C for 45 sec annealing 55 0C for 115 min and
extension at 72 0C for 45 sec gave very good amplification (Fig47)
Fig46 Amplification of the gene at different temperatures
Fig47 Amplification of the gene by optimised PCR conditions
Lane 1 PCR amplification at 50 0C
Lane 2 PCR amplification at 55 0C
Lane 3 PCR amplification at 60 0C
Lane 4 PCR amplification at 65 0C
Lane 5 PCR amplification at 70 0C
Lane 6 PCR amplification using
touchdown PCR
Lane 1 100 bp marker
Lane 2 3 4 PCR products
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
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Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
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aerobic stability of DDT in soil Soil Science Society of America
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
254
452 Large batch amplification and purification of the PCR product
Large batch trails were done to get good quantity of amplified product
The PCR conditions were maintained the same as described under section
4453 A PCR product was run in preparatory agarose gel The amplified
PCR product band was cut and separated from the gel and was purified using
PCR product purification kit (GE health care)
453 Transformation of competent cells
The PCR product was ligated to pTZ57RT vector at 4 0C for 24 h as
per instructions provided along with the kit E coli DH5α grown overnight in
Luria Bertoni broth was treated with CaCl2 to give competent cells The
ligated pTZ57RT plasmid vector containing DDT- dehydrohalogenase gene
was transformed into competent cells of E coli DH5α
The transformants were plated on Luria Bertoni agar containing
ampicillin (100 microg mL) (Fig48) The transformation efficiency was around
50 A total of about 50 transformant colonies that grew on Luria Bertoni-
ampicillin plates were replica plated on to minimal media- DDT- agar
ampicillin plates by toothpick method to select DDT dehydrohalogenase
clones 20 of these colonies grew on minimal media- DDT- agar ampicillin
plates These colonies showed good growth in minimal media- DDT- agar
ampicillin plates
Fig48 Transformant cells on Luria Bertoni- ampicillin agar
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
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Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
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Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
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pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
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intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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element IS1247 Journal of Bacteriology 177 1348- 1356
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
255
454 Selection of the transformants
The colonies that showed good growth in minimal media- DDT- agar
ampicillin plates were then picked up and inoculated to Luria Bertoni broth
containing ampicillin (100 microg mL) The transformants showing maximum
growth in the medium were selected for further studies The transformants
from the medium were harvested and the plasmids were isolated from the
transformants by alkali lysis method (443)
The agarose gel electrophoresis of the transformed plasmids run along
with untransformed pTZ57RT plasmid showed that one of the colonies (E
coli DH- pTZ15) showed an increase in molecular weight indicating the
presence of the complete insert (lane 5 Fig49) The insert in the
transformed clone (E coli DH- pTZ12) was sequenced
Fig49 Gel showing the transformed plasmids
The sequence analysis of Pseudomonas putida T5 indicated that the
protein had 238 residues This amounted to a reverse translation product of
714 base sequences of most likely codons The molecular weight of the
reverse translated protein has been given as 277893 with a theoretical pI of
921
The ExPASSy blast results for finding out the homology of DDT- dhl 1
gene with Pseudomonas spp genome indicated that the sequence had 24-
1 2 3 4 5 6 7
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
256
26 identity with Pseudomonas putida genome The DDT-dhl 1 gene
showed homology with acetolactate synthase gene sequence The southern
blot analysis of genomic DNA from Pseudomonas putida for the DDT- dhl 1
probe was done The genomic DNA of Pseudomonas putida yielded positive
hybridisation More than 1 hybridising bands were observed for few
restriction enzymes digested EcoR1 yielded around 5 bands whereas others
yielded only 2 bands The cloned insert was sub- cloned into pUC18 and
pET28a vectors The expressed protein was analysed for enzyme activity
product of enzyme action and western blot analysis The colorimetric assay
is shown in Fig410 The enzyme exhibited colour change of the dye
because of chloride release The DDT- dehalogenase positive colonies also
exhibited a clearing zone on minimal medium- DDT- agar plates (Fig411)
DDT precipitation disappeared in 5 days because of the degradation of the
compound The western blot analysis gave positive colour with the cloned
protein (Fig412) The product of analysis of the enzyme action indicated
formation of DDD as identified by GC- MS analysis (Fig413)
Fig410 Colorimetric assay of DDT dehydrohalogenase enzyme
Red colour Control Yellow colour DDT- dehydrohalogenase positive
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
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Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
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2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Biological Chemistry 273 33572- 33579
Chapter 2
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Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
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de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
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evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
257
Fig411 DDT-dehydrohalogenase positive plates
Fig412 Western blot hybridization of DDT-dhl1 protein of
Pseudomonas putida probed with antibody raised
against DDT-dehydrohalogenase enzyme
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
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Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
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Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
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pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
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intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
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269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
258
Fig413 GC- MS pattern
A DDT
B DDD
The DDT dehydrohalogenase gene from the clone E coli DH- pTZ15
was recovered by restriction digestion and was ligated to restriction digested
pUC18 cloning vector The transformants were selected as given above
The insert was recovered from the cloned vector by restriction digestion and
this was transformed into pET28a vector This transformed plasmid vector
was cloned into E coli BL 21
The purified PCR product of Pseudomonas putida T5 was labelled
using non radio active nucleic acid labelling by DIG- labelling kit system
according to users guide as described above The genomic DNA of
Pseudomonas putida T5 was restriction digested with Sau 3A Bgl II Sal I
Bam HI Xho I Hind III Pvu II restriction enzymes using appropriate buffers
and incubating for 16hrs at 37 0C The restricted fragments were separated
on 1 agarose gel and transferred on to Nylon membrane The DDT-DHL
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
259
probes were allowed to hybridize with the denatured DNA on the nylon
membrane The southern hybridization of genomic DNA from the strain
Pseudomonas putida T5 revealed the hybridizing DNA fragments for the DDT-
DHL probes tested More than one hybridizing band was observed for few
restriction enzymes used for digestion (Fig414) These results indicated that
presence of more than one band may be due to the presence of more than
one copy of the gene
Fig414 Southern blot hybridization of genomic DNA
of Pseudomonas putida T5 probed with DIG
labelled T5 probe
46 Discussion
The recalcitrance of many synthetic chemicals to biodegradation is
mainly due to the lack of enzymes that can carry out critical steps in the
catabolic pathway This especially holds good for low- molecular weight
halogenated compounds These xenobiotic chemicals are less water soluble
and less bio- available Theoretically these could be converted by short
metabolic routes to intermediates that support cellular growth under aerobic
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
260
conditions Yet no organisms have been found that oxidatively degrade and
use these important environmental chemicals as a carbon source Attempts
to obtain enrichments or pure cultures that aerobically grow on these
chemicals have met no success However some other halogenated
chemicals are easily biodegradable and cultures that utilize chloroacetate 2-
chloropropionate and 1-chlorobutane can be readily enriched from almost any
soil sample (Leisinger 1996 van Agteren et al 1998) For still other
compounds degradative organisms have been isolated but only after
prolonged adaptation due to pre- exposure to halogenated chemicals in the
environment In our studies also a DDT-degrading microbial consortium was
isolated after a long enrichment of the DDT- contaminated soil All the
individual isolates ie ten bacterial strains that constituted the consortium
were found to be necessary for the complete degradation of DDT These
individual isolates were found to act synergistically during degradation of
DDT
With halogenated compounds an obvious critical step in a potential
biodegradation pathway is dehalogenation Biochemical research with
organisms that grow on halogenated compounds has shown that a broad
range of dehalogenases exists both for aliphatic and aromatic compounds
The first enzyme that is responsible in the dehalogenation of DDT to DDD has
been identified as DDT-dehydrodehalogenase (DDT- dhl 1) So far there are
hardly any reports on bacterial DDT-dehydrodehalogenase (DDT- dhl 1)
enzyme and genes encoding the enzyme An attempt was made in our
laboratory to isolate and clone bacterial DDT-dehydrodehalogenase (DDT-
dhl 1) Pseudomonas putida T5 was chosen after preliminary screening by
gradient PCR and touchdown PCR The PCR amplified gene product was
cloned and sequenced The gene showed sequence similarity of 93 to
Acetolactate synthase of Serratia spp There are many xenobiotic
degrading enzymes which have shown such sequence similarities to other
family of enzymes An interesting type of aliphatic dehalogenase is the
enzyme (LinA) that is responsible for the first step in the bacterial degradation
of lindane (-hexachlorocyclohexane) where HCl was eliminated converting
the substrate to pentachlorocyclohexene (Nagata et al 2001 Trantirek et
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
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Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
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Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
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Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
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Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
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Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
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Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
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267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
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Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
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Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
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Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
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Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
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pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
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cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
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268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
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Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
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Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
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Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
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Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
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Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
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Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
261
al 2001) The structure has not been solved but a mechanism was
predicted on the basis of the stereochemistry of the reaction and low but
significant sequence similarity to scytalone dehydratase Similarly hydrolytic
dehalogenase degrading chlorobenzoate (CbzA) (Benning et al 1998) has
been shown to belong to the enoyl hydratase superfamily A specific
hydrolytic dehalogenase (AtzA) involved in the bacterial degradation of
atrazine has been shown to be related to melamine deaminase (TriA)
(Seffernick et al 2001) Dichloromethane dehalogenase (DcmA) catalysing
the conversion of dichloromethane to formaldehyde in a glutathione-
dependent reaction and another group of dehalogenating proteins
chloroacrylic acid dehalogenases (CaaD) which are present in bacteria that
degrade the nematocide 13-dichloropropene have been found to belong to
the tautomerase superfamily of proteins Halohydrin dehalogenases belong
to short- chain dehydrogenase reductase (SDR) superfamily of proteins (de
Jong et al 2003) Alkane hydrolase (AlkB) belongs to a large superfamily of
proteins that also includes non- haem integral membrane desaturases
epoxidases acetylenases conjugases ketolases decarbonylases and
methyl oxidases Atrazine chlorohydrolase (AtzA) belongs to the
amidohydrolase superfamily Other members of the amidohydrolase
superfamily are triazine deaminase hydantoinase melamine deaminase
cytosine deaminase and phosphotriesterase Rhodococcus haloalkane
dehalogenase (DhaA) the dehalogenase gene was preceded by the same
invertase gene sequence and a regulatory gene and on the downstream side
an alcohol dehydrogenase and an aldehyde dehydrogenase encoding gene
(Poelarends et al 2000b)
Analysis of the genetic organization of biodegradation pathways
provides insight into the genetic processes that led to their evolution It
appears that catabolic genes for xenobiotic compounds are often associated
with transposable elements and insertion sequences They are also
frequently located on transmissible plasmids One striking example of a
mobile element that has assisted catabolic genes in their dissemination is IS
1071 This insertion element flanks the haloacetate dehalogenase gene
dehH2 on plasmid pUO1 in Moraxella spp strain B (Kawasaki et al1992)
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
262
the haloalkane dehalogenase gene dhaA on the chromosome in P
pavonaceae 170 (Poelarends et al 2000a) the atrazine degradative genes
atzA atzB and atzC on plasmid pADP-1 in Pseudomonas spp ADP (Wackett
2004) the aniline degradative genes on plasmid pTDN1 in Pseudomonas
putida UCC22 (Fukumori and Saint 1997) and presumably also the p-
sulfobenzoate degradative genes on plasmids pTSA and pPSB in
Comamonas testosterone strains T- 2 and PSB- 4 respectively (Junker and
Cook 1997) These observations clearly indicate that gene mobilization
between and within replicons is an important process during genetic
adaptation It also suggests that genes that are involved in biodegradation of
xenobiotics were recruited from a lsquopre- industrialrsquo gene pool by integration
transposition homologous recombination and mobilization Association of
dehalogenase sequences with mobile genetic elements has also been
observed in other cases viz with haloacetate dehalogenases (Slater et al
1985 Thomas et al 1992 van der Ploeg et al 1995) with dichloromethane
dehalogenase (Schmid-Appert et al 1997) and with -
hexachlorocyclohexane dehalogenase (Dogra et al 2004) In our studies
the native plasmid did not show amplification of the dehalogenase sequence
indicating that the DDT-dehydrohalogenase gene is a genomic character but
not a plasmid borne character
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
Less is known about the origin of the structural genes that encode
critical enzymes such as dehalogenases and the degree of divergence that
occurred during evolution of the current sequences Theoretically it is
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
263
possible that a current gene for a specific critical (dehalogenase) reaction was
already present in the pre-industrial gene pool Alternatively there could be a
short evolutionary pathway that led from an unknown pre- existing gene to the
gene as we currently find it in a biodegradation pathway It has even been
suggested that a new sequence for an enzyme acting on a synthetic
compound could evolve through the activation of an unused alternative open
reading frame of a pre-existing internal repetitious coding sequence (Ohno
1984) Here it should be noted that the similarity of a dehalogenase to
members of an enzyme superfamily that catalyse other reactions generally
does not provide information about the process of adaptation to xenobiotic
compounds The level of sequence similarity that exists between a
dehalogenase and other proteins in a phylogenetic family is usually less than
50 Therefore the time of divergence should be much earlier than a
century ago and the process of divergence thus cannot be related to the
introduction of industrial chemicals into the environment If the
dehalogenases and other critical enzymes that occur in catabolic pathways
have undergone recent mutations there should be closely related sequences
in nature that differ from the current enzymes by only a few mutations No
such primitive dehalogenase has yet been detected with the notable
exception of TriA the enzyme that dehalogenates the herbicide atrazine
Another issue is the function of the pre-industrial dehalogenase or
dehalogenase-like sequences from which the current catabolic systems with
their activated and mobilized genes originate The original genes may have
been involved in the dehalogenation of naturally occurring halogenated
compounds of which there are many (Gribble 1998) Such proteins may
fortuitously also have been active with a xenobiotic halogenated substrate
just because of their lack of substrate specificity Alternatively the
evolutionary precursor of a dehalogenase may have catalysed a different
reaction that has some mechanistic similarity to dehalogenation in which
case the original enzyme may or may not have shown some dehalogenation
activity due to catalytic promiscuity Finally the precursor genes for
dehalogenases may have been silent or cryptic genes with no clear function
for the pre- industrial host (Hall et al 1983) In all cases the gene could
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
264
have become functional in a dehalogenation pathway as a result of the
fortuitous ability to catalyse dehalogenation of a xenobiotic compound
possibly after acquisition of some mutations
One way to obtain information about the evolutionary origin of
dehalogenase genes is to compare the dehalogenase sequences that have
been detected in different bacterial cultures If closely related sequences are
present this would make it possible to identify sequence differences and to
determine the effect of the mutations on substrate selectivity Currently the
whole sequence of more than 300 bacterial genomes is available If we find
closely related sequences in these databases they could define evolutionary
ancestors of the current dehalogenases
When searching for putative alkane hydroxylase genes in
environmental DNA (Venter et al 2004) a large number of alkB and alkM
homologues were again detected including two sequences that were 82
similar to alkB
Even though the same dehalogenase sequences are detected in
organisms that are isolated in different geographical areas and in some
cases even on different substrates they are not the most abundant
dehalogenase sequences identified in whole genome sequencing projects
and massive random sequencing Thus it appears that enrichment
techniques explore a different segment of sequence space than massive
sequencing of environmental DNA The presence of large numbers of
unexplored functional sequences in genomic databases suggests that the
biotransformation scope of microbial systems has an enormous potential for
further growth
47 Conclusions
Bacterial dehalogenases catalyse the cleavage of carbon-halogen
bonds which is a key step in aerobic mineralization pathways of many
halogenated compounds that occur as environmental pollutants DDT-
dehydrohalogenase catalyses the dehydrohalogenation of DDT to DDD with
the removal one HCl The gene responsible for the dehalogenation was
cloned and sequenced The gene sequence showed similarity to acetolactate
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
265
synthase gene There is a broad range of dehalogenases which can be
classified in different protein superfamilies and have fundamentally different
catalytic mechanisms Identical dehalogenases have repeatedly been
detected in organisms that were isolated at different geographical locations
indicating that only a restricted number of sequences are used for a certain
dehalogenation reaction in organohalogen-utilizing organisms
One of the striking observations concerning the distribution and
evolution of key catabolic genes is that identical sequences have repeatedly
been detected in organisms that are enriched on xenobiotic halogenated
substrates as a carbon source Probably the number of solutions that nature
has found to degrade these compounds is very small and horizontal
distribution occurs faster than generation of new pathways Indeed the
dehalogenase genes are often associated with integrase genes invertase
genes or insertion elements and they are usually localized on mobile
plasmids
It is likely that ongoing genetic adaptation with the recruitment of silent
sequences into functional catabolic routes and evolution of substrate range by
mutations in structural genes will further enhance the catabolic potential of
bacteria toward synthetic organohalogens and ultimately contribute to
cleansing the environment of these toxic and recalcitrant chemicals
References
Ahmed D Sylvester M Sondossi M 1991 Subcloning of bph genes from
Pseudomonas testosteroni B-356 in Pseudomonas putida and
Escherichia coli evidence from dehalogenation during initial attack on
chlorobiphenyl Applied and Environmental Microbiology 57 2880-
2887
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
266
Bourquin AW 1977 Degradation of malathion by salt- marsh
microorganisms Applied and Environmental Microbiology 33 356-
362
Chacko CL Lockwood JL Zabik M 1966 Chlorinated hydrocarbon pesticides
degradation by microbes Science 154 893- 895
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Dogra C Raina V Pal R Suar M Lal S Gartemann KH 2004 Organization
of lin genes and IS6100 among different strains of
hexachlorocyclohexane-degrading Sphingomonas paucimobilis
evidence for horizontal gene transfer Journal of Bacteriology 186
2225- 2235
Fukumori F Saint CP 1997 Nucleotide sequences and regulational analysis
of genes involved in conversion of aniline to catechol in Pseudomonas
putida UCC22 (pTDN1) Journal of Bacteriology179 399- 408
Gribble GW 1998 Naturally occurring organohalogen compounds Acc
Chemical Research 31 141- 152
Guenzi WD Beard WE 1968 Anaerobic conversion of DDT to DDD and
aerobic stability of DDT in soil Soil Science Society of America
Proceedings 32 522- 527
Hall BG Yokoyama S Calhoun DH 1983 Role of cryptic genes in microbial
evolution Molecular Biology and Evolution 1 109- 124
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
267
Junker F Cook AM 1997 Conjugative plasmids and the degradation of
arylsulfonates in Comamonas testosterone Applied Environmental
Microbiology 63 2403- 2410
Kawasaki H Tsuda K Matsushita I Tonomura K 1992 Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid
from Moraxella sp strain B Journal of General Microbiology 138
1317- 1323
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the
head of bacteriophage T4 Nature (London) 227 680- 685
Leisinger T 1996 Biodegradation of chlorinated aliphatic compounds
Current Opinions in Biotechnology 7 295-300
Maniatis T Fritsch EF Sambrook J 1982 Molecular cloning- A laboratory
manual Cold Spring Harbour Laboratory New York
Nadeau LJ Menn FM Breen A Sayler GS 1994 Aerobic degradation of
111-trichloro-22-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes
eutrophus A5 Applied and Environmental Microbiology 60 51- 55
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma-
hexachlorocyclohexane dehydrochlorinase LinA Proteins 45 471-
477
Ohno S 1984 Birth of a unique enzyme from an alternative reading frame of
the preexisted internally repetitious coding sequence Proceedings of
National Academy of Sciences USA 81 2421- 2425
Poelarends GJ Kulakov LA Larkin MJ van Hylckama Vlieg JET Janssen
DB 2000a Roles of horizontal gene transfer and gene integration in
evolution of 1 3 dichloropropene- and 1 2 dibromoethane- degradative
pathways Journal of Bacteriology 182 2191- 2199
Poelarends GJ Zandstra M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
268
Ramos JL Diacuteaz E Dowling D Lorenzo V Molin S Gara F Ramos C Timmis
KN 1994 The behavior of bacteria designed for biodegradation
Biotechnology 12 1349- 1356
Ridder IS Rozeboom HJ Kalk KH Dijkstra BW 1999 Crystal structures of
intermediates in the dehalogenation of haloalkanoates by L-2-haloacid
dehalogenase Journal of Biological Chemistry 274 30672- 30678
Schmid-Appert M Zoller K Traber H Vuilleumier S Leisinger T 1997
Association of newly discovered IS elements with the dichloromethane
utilization genes of methylotrophic bacteria Microbiology 143 2557-
2567
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Singh BK Kuhad RC Singh A Lal R Tripathi KK 1999 Biochemical and
molecular basis of pesticide degradation by microorganisms Critical
Reviews in Biotechnology 19(3)197- 225
Slater JH Weightman AJ Hall BG 1985 Dehalogenase genes of
Pseudomonas putida PP3 on chromosomally located transposable
elements Molecular Biology and Evolution 2 557- 567
Thomas AW Slater JH Weightman AJ 1992 The dehalogenase gene dehI
from Pseudomonas putida PP3 is carried on an unusual mobile genetic
element designated DEH Journal of Bacteriology 174 1932- 1940
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
van Agteren MH Keuning S Janssen DB 1998 Handbook on
Biodegradation and Biological Treatment of Hazardous Organic
Compounds Dordrecht the Netherlands Kluwer Academic Publishers
van der Ploeg J Willemsen M van Hall G Janssen DB 1995 Adaptation of
Xanthobacter autotrophicus GJ10 to bromoacetate due to activation
and mobilization of the haloacetate dehalogenase gene by insertion
element IS1247 Journal of Bacteriology 177 1348- 1356
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
269
Venter JC Remington K Heidelberg JF Halpern AL Rusch D Eisen JA
2004 Environmental genome shotgun sequencing of the Sargasso
Sea Science 304 66- 74
Wackett LP 2004 Evolution of enzymes for the metabolism of new chemical
inputs into the environment Journal of Biological Chemistry 279
41259- 41262
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl)ethane by Aerobacter aerogenes Applied Microbiology
15 569- 574
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
270
4A1 Introduction
The biological destruction of toxic and hazardous chemicals is an
important contribution of microbial metabolism Microorganisms convert
complex hazardous organic compounds via their central metabolic routes to
CO2 or other simple organic compounds Organisms that bring about their
degradation have been isolated from their natural environments During
aerobic degradation of chlorinated compounds by microorganisms molecular
oxygen serves as the electron acceptor Several chloroaliphatic compounds
have been shown to be degraded aerobically Microbes play an essential role
in the bioconversion and total breakdown of pesticides Among the microbial
communities bacteria and fungi are the major degraders of pesticides
Yeasts microalgae and protozoa are less frequently encountered in the
degradation process
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer 1967) The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT with the formation of DDD (11-dichloro-
22-bis(4-chlorophenyl) ethane or dichlorodiphenyldiichloroethane) (Kallman
and Andrews 1963 Barker and Morrison 1964) Under aerobic conditions the
first product of DDT metabolism has been found to be DDD by
dehydrochlorination DDD was further degraded through dechlorination
dehydrochlorination and decarboxylation to DBP or to a more reduced form
DDM
In our laboratory DDT-dehydrohalogenase of Pseudomonas putida T5
was shown to be converted to DDD The enzyme protein was purified and
characterized An attempt was made to study the binding pattern of
substrates and inhibitors of DDT-dehydrohalogenase using a computational
method Threendashdimensional models were constructed and substrate
specificity binding site of enzymes and the activity of the selected substrates
were compared using the molecular docking A comparison of theoretical and
experimental results of enzyme activity was also investigated
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
271
4A2 Materials and methods
4A21 Softwares
The following softwares were used in molecular modelling of DDT-
dehydrohalogenase
4A211 BLAST computer programme
Similarity to the DDT-dehydrohalogenase nucleotide sequence was
obtained by using BLAST computer programme
4A212 Clustal W
Multiple sequence alignment was done using Clustal W
4A213 COMPUTE PI
Primary structure analysis tools such as COMPUTE PI was used for
isoeletric point and molecular weight
4A214 PROTPARAM
This was used for deciphering computation of various physical and
chemical parameters for a given protein sequence
4A215 Rapid automatic Detection and alignment of repeats (Radar)
Rapid automatic Detection and alignment of repeats in protein
sequences programme is used for alignment of repeats in protein sequences
4A216 MOTIFS
MOTIFS is to identify the number of motifs in a sequence
4A217 CONSERVED DOMAIN DATABASE
This is used to identify the conserved domain present in a protein
sequence
4A218 PROSITE
This is to scan a protein sequence for the occurrence of patterns and
profiles stored in the prosite data
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
272
4A129 PEPINFO
This is used to plot simple amino acids properties in parallel and Plot
of hydrophobicity
4A2110 PEPWINDOW
This will display protein hydropathy
4A2111 PEPSTATS
This was used to report information on Molecular Weight Number of
residues Average residue weight Charge Isoeletric point for each physic
chemical class of aminoacids and Molar extinction coefficient at 1mgmL
4A2112 Peptide Cutter
This predicts potential cleavage sites cleaved by proteases or
chemicals in a given Protein sequence
4A2113 Reverse Translate
Reverse Translate accepts a protein sequence as input and uses a
codon usage table to generate a DNA sequence representing the most likely
non-degenerate coding sequence
4A2114 Neural Network
Neural network was used to predict the secondary structure
4A2115 3DPSSM OR PHYRE
3DPSSM OR PHYRE was used to predict the Tertiary structure
4A2117 Discovery studio 20
Docking was processed by Discovery studio 20
Homology modelling
Structural modelling of the sequence of DDT-dehydrohalogenase
obtained from the above sequencing process (Uniprot
tr|D0QB56|D0QB56_PSEPU DDT dehydrohalogenase) was done using the
Phyre2 remote homology modelling server since the conventional BLAST
program could not find homologues with satisfying sequence similarity and
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
273
structure Phyre protocols can be used to achieve high accuracy models at
very low sequence identities (15ndash25) (Kelley LA and Sternberg MJE 2009)
The query sequence was submitted to the Phyre2 server The Phyre2 server
uses a library of known protein structures taken from the Structural
Classification of Proteins (SCOP) database (Alexey et al 1995) The
sequence of each of these structures is scanned against a non-redundant
sequence database and a profile constructed and deposited in the lsquofold
libraryrsquo The query sequence is scanned against the non-redundant
sequence database and a profile is constructed Five iterations of PSI-Blast
are used to gather both close and remote sequence homologs and are
combined into a single alignment with the query sequence as the master
Following profile construction the query secondary structure is predicted
Three independent secondary structure prediction programs are used in
Phyre Psi-Pred (McGuffin et al 2000) SSPro (Cheng et al 2005) and JNet
(Cuff etal 1999) These programs provide a confidence value at each
position of the query for each of the three secondary structure states These
confidence values are averaged and a final consensus prediction is
calculated In addition the program Disopred (Ward et al 2004) is run to
calculate a two-state prediction of which regions of the query are most likely
to be structurally ordered (o) and which disordered (d) This profile and
secondary structure is then scanned against the fold library using a profilendash
profile alignment algorithm detailed in Bennett-Lovsey et al 2008 This
alignment process returns a score on which the alignments are ranked An e-
value is calculated by fitting the scores to an extreme value distribution The
top ten highest scoring alignments are then used to construct full 3D models
of the query Where possible missing or inserted regions caused by
insertions and deletions in the alignment are repaired using a loop library and
reconstruction procedure Finally side- chains are placed on the model using
a fast graph-based algorithm and side chain rotamer library Phyre2 also
incorporates ab initio folding simulation called Poing2 to model regions of the
proteins with no detectable homology to known structures The model
generated by Phyre2 is then retrieved and the quality of the model was
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
274
evaluated Profiles- 3D and assessed using Ramachandran map using
Accelrys Discovery studio 25 (DS25)
4A2118 CDOCKER
CDOCKER is an implementation of a CHARMm based docking tool
using a rigid receptor
The following steps are included in the CDOCKER protocol
A set of ligand conformations were generated using high-temperature
molecular dynamics with different random seeds
Random orientations of the conformations were produced by
translating the center of the ligand to a specified location within the receptor
active site and performing a series of random rotations A softened energy
was calculated and the orientation was kept if the energy was less than a
specified threshold This process was continued until either the desired
number of low-energy orientations were found or the maximum number of
bad orientations was tried
Each orientation was subjected to simulated annealing molecular
dynamics The temperature was heated up to a high temperature then cooled
to the target temperature
A final minimization of the ligand in the rigid receptor using non-
softened potential was performed
For each final pose the CHARMm energy (interaction energy plus
ligand strain) and the interaction energy alone were calculated The poses
were sorted by CHARMm energy and the top scoring (most negative thus
favorable to binding) poses were retained
4A3 Results and Discussion
4A31 Sequence analysis
The sequence of DDT-dehydrohalogenase along with amino acid
sequence is given in the fig4A31
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
275
Fig4A31 Sequence analysis of DDT-dehydrohalogenase
The enzyme was found to have 238 amino acid residues (Table
4A31)
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
276
4A32 Sequence similarity by BLAST searching
DDT-dehydrohalogenase was found to have substantial sequence
similarity to that of acetolactate synthase of few bacterial strains The N-
terminal and C-terminal homology is given in fig4A32a and fig4A32b
Db AC Description Score E-value
tr Q4K606 _PSEF5 SubName Full=Acetolactate synthase larg 33 0029
tr Q4K6F7 _PSEF5 SubName Full=Acetolactate synthase II l 33 0038
tr Q4K790 _PSEF5 SubName Full=Acetolactate synthase larg 32 0064
tr Q4KG06 _PSEF5 SubName Full=Glyoxylate carboligase 27 21
tr Q4K9C1 _PSEF5 SubName Full=Cyclic beta 1-2 glucan synt 27 21
tr Q4K7N6 _PSEF5 SubName Full=Putative uncharacterized pr 26 35
Fig4a31a Graphical overview of the alignments
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
277
Alignments
tr Q9HVA0 Q9HVA0_PSEAE
SubName Full=Acetolactate synthase large subunit [Name=ilvI] [Pseudomonas aeruginosa]
574 AA
Score = 381 bits (87) Expect = 8e-04 Identities = 32103 (31) Positives = 49103 (47) Gaps = 7103 (6) Query 6 HNLDIKIILMNNQALGMVHQQQTLMFNEHIVASAYPYQTD--TIAKGF---GLHTCDLNK 60 ++L +KI+ +NN ALGMV Q Q + +N S D +A+ + G+ DL K Sbjct 466 YDLPVKIVNLNNGALGMVRQWQDMQYNSRYSHSYMESLPDFVKLAEAYGHVGMRITDL-K 524 Query 61 DSDPHAALQAAIERPGPCLIHALIDVSEKVWPMVLPGDANIDM 103 D P +A + + +D SE V+PM + A DM Sbjct 525 DLKPKME-EAFAMKNRLVFLDIQVDASEHVYPMQIRDGAMRDM 566
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
278
Fig4A32a Multiple sequence alignment of N terminal
region of homologous
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
279
Fig4A32b Multiple sequence alignment of C terminal
region of homologous
4A33 Composition of the enzyme
Number of amino acids 238
Molecular weight 277893
Theoretical pI 921
Total number of negatively charged residues (Asp + Glu) 29 (Table 4A31)
Total number of positively charged residues (Arg + Lys) 44
Grand average of hydropathicity (GRAVY) -053
The amino acid with highest composition is lysine and the protein was found
to be stable
Grand average of hydropathicity (GRAVY) -0536
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
280
Table 4A31 Amino acid composition of DDT-dehydrohalogenase
Amino acid Amino acid Amino acid
Ala (A) 19 80
80 Ile (I) 10 42 Tyr (Y) 11 46
Arg (R) 17 71 Leu (L) 14 59 Val (V) 13 55
Asn (N) 7 29 Lys (K) 27 113 Pyl (O) 0 00
Asp (D) 15 63 Met (M) 9 38 Sec (U) 0 00
Cys (C) 14 59 Phe (F) 12 50 (B) 0 00
Gln (Q) 7 29 Pro (P) 9 38 (Z) 0 00
Glu (E) 14 59 Ser (S) 11 46 (X) 0 00
Gly (G) 11 46 Thr (T) 8 34
His (H) 5 21 Trp (W) 5 21
Lysine was 113 followed by alanine (8) Histidine and tryptophan were
low in concentration (21)
4A34 Atomic composition
Carbon C 1234
Hydrogen H 1927
Nitrogen N 345
Oxygen O 341
Sulfur S 23
Formula C1234H1927N345O341S23
Total number of atoms 3870
4A35 Extinction coefficients
Extinction coefficients are in units of M-1 cm-1 at 280 nm measured in water
Ext coefficient 44765
Abs 01 (=1 gl) 1611 assuming ALL Cys residues appear as half cystines
The estimated half-life is 30 hours
4A36 Instability index
The instability index (II) is computed to be 3624 this classifies the protein as
stable
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
281
4A37 Aliphatic index
Aliphatic index is 6315
4A38 Formula
C1234H1927N345O341S23 and total number of amino acid in the given
sequence is 238
4A39 RADAR RESULTS
RADAR did not find any repeats in the sequence
4A310 MOTIF RESULTS
Number of found motifs 112
No motif was found in prosite pattern
No motif was found in prosite profile
4A311 CD DOMAIN RESULTS
Fig4A33a CD DOMAIN RESULTS
10 20 30 40 50 60 70
80
||||||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
GEKHLCDKFAKILR 43
ARDDEKWLWMMHDTRAVCAIFqYGKVWQRPKLIMTRVDAE-
AEEYKQKMEDILDFNEKSYTGGGcsKPLTGCEIkSTADF 121
cd03183 3
ARVDEYLAWQHTNLRLGCAKY-
FWQKVLLPLLGGKPVSPEkVKKAEENLEESLDLLENYFLKDK--
PFLAGDEI-SIADL 78
90 100 110 120
||||
MAIGRVHNAKSKLRYKFDLMSPSRELYIFLESRTKQPYECKCVNLG
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
282
GEKHLCDKFAKILR 122 GAKFENEPKMAGFRNCCVGKP-
MTAWKARVRAKICNCKYQAHKRL 165
cd03183 79
SAVCEIMQPEAAGYDVFEGRPkLAAWRKRVKEAGNPLFDEAHKII
123
Fig4A33b Hphob Kyte amp Doolittle Plot
Property Residues Number Mole
Tiny (A+C+G+S+T) 63 26471
Small (A+B+C+D+G+N+P+S+T+V) 107 44958
Aliphatic (I+L+V) 37 15546
Aromatic (F+H+W+Y) 33 13866
Non-polar (A+C+F+G+I+L+M+P+V+W+Y 127 53361
Polar (D+E+H+K+N+Q+R+S+T+Z) 111 46639
Charged (B+D+E+H+K+R+Z) 78 32773
Basic (H+K+R) 49 20588
Acidic (B+D+E+Z) 29 12185
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
283
4A312 Secondary Structure Analysis
Fig4A34 Secondary Structure Prediction
The protein structure belongs to a β fold The structure has 12 β-
sheets 9 long and 3 short (Fig4A34 amp Fig4A35)
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
284
Fig4A35 Predicted secondary structure and the
disorder prediction of the protein
Homology modelling
BLAST search against the structure database could find only two
models with sequence identity of only around 27 Homology modelling of
the newly purified protein DDT halogenase Pseudomonas putida T5 (Uniprot
tr|D0QB56|D0QB56_PSEPU) was done using remote homology modelling
server Phyre2 BLAST search against the sequence database the predicted
GST-N-Theta sub family domain in the region of residues 2-68 and a GST-C-
Theta subfamily in the region of residues 83-161 The GST fold contains an
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
285
N-terminal thioredoxin-fold domain and a C-terminal alpha helical domain
with an active site located in a cleft between the two domains GSH binds to
the N-terminal domain while the hydrophobic substrate occupies a pocket in
the C-terminal domain Class Theta subfamily comprises of eukaryotic class
Theta GSTs and bacterial dichloromethane (DCM) dehalogenase (Marchler-
Bauer A et al 2011) Fig4A34 shows the secondary structure and the
disorder prediction of the protein as predicted by Phyre2 The protein
structure belongs to β fold DDT-dehydrohalogenase is composed of 8
helices and has 4 β-sheets inter-attached by random coils Phyre2 server
predicted the model by aligning regions of domains of PDB structures
c3c8eB c1ljrB 3 c2c3nB 4 c2x64A c3nivD c2jl4A c2fnoB c3lykA
c1zl9A c1f2eB c3h1nA c2ntoA c3ergA c1r5aA c2pmtA c1ua5A
c1nhyA c3lszA c3uarA and c1k3yB these structures were aligned with
100 confidence and the percentage of identity were between 12-24
PSIPRED graphical out put from prediction of DDT-
dehydrohalogenase produced by PSIPRED view
PSIPRED ndashprotein structure prediction server incorporates three
recently developed methods PSIPRED GenTHREADER and MEMSAT 2 for
predicting structural information about a protein from its amino acid sequence
alone PSIPRED (Jones 1999) carried out reliable secondary structure
prediction on a protein The DDT-dehydrohalogenase is composed of 4
helices 12 β-sheets interattached by random coils Fig4A37 amp Fig4A38
show the 3D modelling of the enzyme
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
286
Fig4A36 Hydrophobic index of DDT-dehydrohalogenase
+0531
-0103
1 200
Hydrophobicity
indices at
ph 34
determined
by HPLC
+0827
-082
0
1 200
Hydropathicity
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
287
4A313 3D structure analysis
Fig4A37 3D structure viewed in Accelrys DS Visualizer Software
Stereo view of Cartoon representation of DDT-
dehydrohalogenase enzyme
A B
C D
A is Wire model B is Ribbon model showing β sheets
C is Barallel model D is Ribbon model showing α helices
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
288
Fig4A38 Ribbon model of the modelled protein generated by Phyre2
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
289
Fig4A39 Ramachandran plot
Ramachandran plot developed by Gopalasamudram Narayana
Ramachandran is a way to visualize dihedral angles φ against ψ of amino
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
290
acid residues in protein structure It shows the possible conformations of φ
and ψ angles for a polypeptide In a polypeptide the main chain N-C alpha
and C alpha-C bonds relatively are free to rotate These rotations are
represented by the torsion angles phi and psi respectively Computer
models were used to systematically vary phi and psi with the objective of
finding stable conformations For each conformation the structure was
examined for close contacts between atoms Atoms were treated as hard
spheres with dimensions corresponding to their van der Waals radii
Therefore phi and psi angles which caused spheres to collide correspond to
sterically disallowed conformations of the polypeptide backbone In the
diagram given above the white areas correspond to conformations where
atoms in the polypeptide come closer than the sum of their van der Waals
radii These regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain The red regions
correspond to conformations where there are no steric clashes ie these are
the allowed regions namely the alpha-helical and beta-sheet conformations
The yellow areas show the allowed regions if slightly shorter van der Waals
radii are used in the calculation ie the atoms are allowed to come a little
closer together This brings out an additional region which corresponds to the
left-handed alpha-helix L-amino acids cannot form extended regions of left-
handed helix but occasionally individual residues adopt this conformation
These residues are usually glycine but can also be asparagine or aspartate
where the side chain forms a hydrogen bond with the main chain and
therefore stabilises this otherwise unfavourable conformation The 3(10) helix
occurs close to the upper right of the alpha-helical region and is on the edge
of allowed region indicating lower stability Disallowed regions generally
involve steric hindrance between the side chain C-beta methylene group and
main chain atoms Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran plot Hence it
frequently occurs in turn regions of proteins where any other residue would be
sterically hindered
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
291
Fig4A310 Ramachandran map of the modeled protein
The plot combines the four separate Ramachandran maps (as shown
to the right) using shapes to distinguish the membership to a particular class
(General Proline Glycine and Pre-Proline) Within this scheme Glycines are
shown as diamonds Prolines as triangles residues preceding Prolines (Pre-
Proline) have a rectangular shape otherwise (General case) they are drawn
as small squares
Ramachandran map of the model calculated using DS25 is shown in
fig4A39 amp fig4A310 9725 percentage of the residues are in the allowed
region residues Ala56 Ile115 Thr142 Cys153 and two glycine are in outlier
region
Before energy minimization in the figure above the white areas
corresponds to conformations where atoms in the polypeptide come closer
than the sum of their van der Waals radii These regions are sterically
disallowed for all amino acids except glycine which is unique in that it lacks a
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
292
side chain The red regions correspond to conformations where there are no
steric clashes ie these are the allowed regions namely the alpha-helical
and beta-sheet conformations The yellow areas show the allowed regions if
slightly shorter van der Waals radii are used in the calculation ie the atoms
are allowed to come a little closer together This brings out an additional
region which corresponds to the left-handed alpha-helix L-amino acids
cannot form extended regions of left-handed helix but occasionally individual
residues adopt this conformation These residues are usually glycine but can
also be asparagine or aspartate where the side chain forms a hydrogen bond
with the main chain and therefore stabilises this otherwise unfavourable
conformation The 3(10) helix occurs close to the upper right of the alpha-
helical region and is on the edge of allowed region indicating lower stability
Disallowed regions generally involve steric hindrance between the side chain
C-beta methylene group and main chain atoms Glycine has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the
Ramachandran plot Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered
After energy minimization the colour scheme encodes the affiliation to
a certain region within the respective Ramachandran Map Residues in the
core region are rendered in green or yellow if they are found to be in the
allowed region and red if in the outlier region Outliers are always marked as
crosses independent of their class
The colour option allows for viewing the plot in either black or white
background colour When White background is selected the above
mentioned colouring scheme for assignment to core allowed or outlier region
within the map is not used instead filled or non-filled shapes are used to
make class
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
293
4A313b Molecular surface analysis
Fig4A311 Surface analysis of DDT-dehydrohalogenase
Using molecular surface analysis active site could be predicted
Hydrophobic region is in dark green colour rose colour indicated hydrogen
bonding region and Blue colour indicated the mid polar region The green
shade is the predicted active site It indicated a hydrophobic core It favours
hydrophobic ligands (Fig4A311)
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
294
4A314 Rotamer strain energy plot
The Plot depicts the rotamer strain calculated energy (kcalmol) The
X-Axis shows the residues in sequential order within MOE Residues with
strain energies larger than 50 kcalmol (marked by the dotted horizontal line
in dark red colour) (Fig4A312) are infrequent in the PDB and warrant a
closer inspection The strain energy threshold of the result set may be
adjusted with the E-Threshold slider reducing the results to entries with strain
energies above the designated cut off
Fig4A312 Rotamer profile of DDT-dehydrohalogenase
4A315 Prediction of active site
Active site was predicted using binding site molecule of discovery studio
software which predicted 6 possible ligand binding sites (active site) Since
the DDT was reported to be ligand for this protein Based on the chemical
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
295
properties of this ligand or DDT we have chosen site no1 as the probable
active site which comprises of
Active site cavity which comprises of His30 Leu25 Gln42 Glu28 Glu
104 Tyr105 Trp 109 Arg 111 Lys 113 Leu 114 and Lys215 were identified
as substrate binding site
4A316 Molecular docking
4A316a Molecular docking of DDT into active site
Fig4A313 Molecular docking of ligand DDT into the active site
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
296
Fig4A314 Interaction of the ligands with the active site
4A316b Docking of ligand DDT along with glutathione into the active Site
The GST active site is composed of a GSH binding site (G-site)
common to all GSTs and a xenobiotic binding site (H-site) which varies
between different classes and isotypes Residues from the N-terminal TRX-
fold domain form the G-site while the H-site is comprised mainly of residues
from the C-terminal alpha helical domain (Marchler-Bauer et al 2011)
In order to comprehend the molecular level interaction of ligands
Glutathione and DDT docking of the co-factor Glutathione was carried out first
followed by docking of DDT to the modelled Glutathione DDT dehalogenase
complex The predicted glutathione binding site was similar to GST- N family
Class Theta subfamily composed of eukaryotic class Theta GSTs and
bacterial dichloromethane (DCM) dehalogenase Reduced Glutathione was
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
297
successfully docked into the predicted binding site which comprised of
residues Ser1 Glu5 Leu25 Glu28 Lys29 His30 Leu31 Arg40 Phe41
Glut42 Leu55 Ser57 Arg97 and Tyr105 In many GSTs activation of
glutathione (GSH) to GS- is accomplished by a Tyr at H-bonding distance
from the sulphur of GSH (Zheng et al 1997 Mannervik et al 2005)
Glutathione makes three hydrogen bond contact with the active site residues
The Sulphur atom of Glutathione is making a hydrogen bond contact with
Tyr105O atom (H-bond distance 304Aring) N1 atom of Glutathione is making a
bisected hydrogen bond with Glu166O2 atom Ser57OG atom is also
making a hydrogen bond contact with O4 atom of Glutathione (h-bond
distance 315 Aring)
GSTs catalyse nucleophilic attack by reduced glutathione (GSH) on
nonpolar compounds that contain an electrophillic carbon nitrogen or sulphur
atom (Zheng et al 1997 Mannervik et al 2005) The nonpolar substrate
DDT was docked into the active site cleft located between the two domains
TRX-fold and the C-terminal alpha helical domain The hydrophobic ligand
DDT is making a number of van der walls contact with the active site G-site
which comprised of residues Leu25 Gly 26 Gly27 Glu28 His30 Leu31
Gln42 Val43 Glu 104 Tyr105 Trp 109 Gln110 Lys215 Pro216 Phe218
and to the docked Glutathione Fig4A313 shows the interaction of the
ligands to the active site residues and the Fig4A314 shows the molecular
surface representation of the docked complex Calculated free energy of
binding was -4062 and -2645 kcalmol for GSH and DDT respectively
The structures of reduced form of Glutathione and DDT were built
using the Build fragment module of DS25 and energy minimised using
standard energy minimisation protocols of DS25 Conserved residues of the
binding site Ser1 (Highly conserved) Leu25 (Highly conserved) Ser57
(Highly conserved) Arg97 (Highly conserved) Lys29 His30 Arg40 were
rendered flexible during the docking simulation of Glutathione (Fig4A315)
(Supariya 2009) CHARMm force field (Brooks et al 1983) was applied to
both the protein and ligand and Monmany Rone force field was used for the
calculation of partial charges Glutathione was flexibly docked into the
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
298
identified glutathione binding site based on the standard flexible docking
protocols implemented in DS25 in which docking refinement was carried out
using Simulated Annealing protocol with 2000 steps of Heating 5000 steps of
cooling Free energy of binding was calculated with the Calculate Free
Energy Binding module with a distance depended di-electric constant as
implicit solvent model and a di-electric constant of 78 The final docked
complex was selected based on orientation of the ligand in respect to other
structural homologs and estimated Free Energy of binding
The Substrate DDT was similarly docked in the identified substrate
binding site of the structure of Glutathione DDT dehalogenase complex DDT
is attached by 2 hydrogen bonds with active site amino acids DDT 2 chloride
(atoms) are attached by hydrogen bond with aminoacids CYS 65H N Bond
distance 229200 A and 5th chloride of DDT is attached by hydrogen bond
with amino acids GLN130 HE21 Bond distance 248 A In this picture
orange colour indicates DDT and yellow colour indicate co factors such as
glutathione (Fig4A316)
Name Neighbour Monitor1
Distance 5000000
Show distances No
Show intermolecular only Yes
Number of Neighbours 418
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
299
Fig4A315 Docking of glutathione as a cofactor into the active site
Fig4A316 Molecular surface representation of the docked complex
ligand DDT on the left and GSH on the right represented as stick
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
300
4A316c Docking of ligand DDT along with glutathione into the active
Site
Glutathione which enhanced the activity of DDT-dehydrohalogenase to
312 folds was docked along with DDT
The structure of a glutathione was built with minimized energy using
builder module of discovery studio This was then docked into the active site
of the modelled structure using c-docker Green dotted line indicates
hydrogen bond with aminoacids in the activesite
Fig4A317 Ligand DDT contact with Wanderwall force
In the Fig4A317 green colour shows wanderwall interaction towards
ligand DDT during enzyme interaction with DDT in Pseudomonas putida T5
The vander waals radii were completely attached with DDT DDT
dehydrohalogenase was assigned a position in the Phylogenetic tree (Fig
4A318)
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
301
Fig 4A318 Phylogenetic tree of DDT-dehydrohalogenase
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
302
4A4 Discussion
The PCR amplified gene product was cloned and sequenced The
gene showed sequence similarity of 93 to Acetolactate synthase of Serratia
spp There are many xenobiotic degrading enzymes which have shown such
sequence similarities to other family of enzymes An interesting type of
aliphatic dehalogenase is the enzyme (LinA) that is responsible for the first
step in the bacterial degradation of lindane (-hexachlorocyclohexane) where
HCl was eliminated converting the substrate to pentachlorocyclohexene
(Nagata et al 2001 Trantirek et al 2001) The structure has not been
solved but a mechanism was predicted on the basis of the stereochemistry of
the reaction and low but significant sequence similarity to scytalone
dehydratase Similarly hydrolytic dehalogenase degrading chlorobenzoate
(CbzA) (Benning et al 1998) has been shown to belong to the enoyl
hydratase superfamily A specific hydrolytic dehalogenase (AtzA) involved in
the bacterial degradation of atrazine has been shown to be related to
melamine deaminase (TriA) (Seffernick et al 2001) Dichloromethane
dehalogenase (DcmA) catalysing the conversion of dichloromethane to
formaldehyde in a glutathione- dependent reaction and another group of
dehalogenating proteins chloroacrylic acid dehalogenases (CaaD) which are
present in bacteria that degrade the nematocide 13-dichloropropene have
been found to belong to the tautomerase superfamily of proteins Halohydrin
dehalogenases belong to short- chain dehydrogenase reductase (SDR)
superfamily of proteins (de Jong et al 2003) Alkane hydrolase (AlkB)
belongs to a large superfamily of proteins that also includes non- haem
integral membrane desaturases epoxidases acetylenases conjugases
ketolases decarbonylases and methyl oxidases Atrazine chlorohydrolase
(AtzA) belongs to the amidohydrolase superfamily Other members of the
amidohydrolase superfamily are triazine deaminase hydantoinase melamine
deaminase cytosine deaminase and phosphotriesterase Rhodococcus
haloalkane dehalogenase (DhaA) the dehalogenase gene was preceded by
the same invertase gene sequence and a regulatory gene and on the
downstream side an alcohol dehydrogenase and an aldehyde dehydrogenase
encoding gene (Poelarends et al 2000)
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
303
It has become clear that most dehalogenases belong to protein
superfamilies that harbour both dehalogenases and proteins that carry out
completely different reactions (de Jong and Dijkstra 2003) Hisano et al
(1996) and Ridder et al (1999) defined the haloalkane dehalogenases
belonging to HAD superfamily of hydrolases Possessing a αβ- hydrolase
fold main domain the α β- hydrolase structural fold is shown also to be found
in lipases acetylcholinesterases esterases lactonases epoxide hydrolases
and others showing that the haloalkane dehalogenases belong to a protein
superfamily of which the members carry out diverse reactions mostly with
non-halogenated compounds
The nucleic acid sequence of Pseudomonas putida T5 was used for
bioinformatics work Based on BLAST search against sequence database
putative conserved domain have been detected The predicted domain is
GST-C-family superfamily It is in Class Theta subfamily composed of
eukaryotic class Theta GSTs and bacterial dichloromethane (DCM)
dehalogenase The GST fold contains the N-terminal thioredoxin-fold domain
and a C-terminal alpha helical domain with an active site located in a cleft
between the two domains GSH binds to the N-terminal domain while the
hydrophobic substrate occupies a pocket in the C-terminal domain Using
compute PI and protparam molecular weight and physical and chemical
properties have been detected using RADAR and prosite repeats and blocks
pfam was detected The identified domains were then designated according
to their pore forming residues The secondary structure was predicted by
nnpredict
For construction of 3D model Pseudomonas putida T5 sequence was
submitted to PSI-BLAST (httpwwwncbinlmnihgovBLAST) The
comparative model failed to find a suitable template for the Pseudomonas
putida T5 sequence Therefore threading method was employed to find an
appropriate template using 3D Position Specific Scorings Matrix server
(3DPSSM or phyre) (httpwwwsbgbioicacuk~3dpssm) The server has
predicted many structures similar to Pseudomonas putida T5 sequence From
these c1v2ab was selected based on their sequence identity and some
logical ideas The sequence identity is 18 and length is 210cm Its family
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
304
and superfamily are Glutathione transferase gst1-6 and glutathione-s-
transferase 1-6 from Anopheilies dirus respectively Therefore a 3D model
was constructed using 3dpssm or phyre The aligned sequence of ppt5 and
c1v2ab was adjusted manually (minimization) to get better structural quality of
the model corresponding to the template structure using DISCOVERY
STUDIO 20 The side chain conformations of the constructed model were
refined to minimize the error using the Side Chain with Rotamer Library
The constructed model of the Pseudomonas putida T5 protein was
evaluated for its backbone conformation (structural quality) using
Ramachandran plot The Ramachandran plot shows the residue backbone
conformations for the modelled protein as well as the template The plot
combines the four separate Ramachandran maps using shapes to distinguish
the membership to a particular class (General Proline Glycine and Pre-
Proline) The Auto Deposition Input Tool (ADIT) available online
(httpdepositpdborgvalidate) was used to check the favourable and
unfavourable properties of the model structure before it was deposited in the
Protein Data Bank Presence of active sites and pockets in the protein
Pseudomonas putida T5 was predicted using binding site molecule of
discovery studio software which predicted six possible ligand binding
The three-dimensional model was constructed for Pseudomonas
putida T5 with the receptor protein and DDT analogues as flexible ligands
The DDT analogues having more than 90 structural similarity with DDT
were retrieved from the PubChem database
(httppubchemncbinlmnihgov) Since DDT was reported to be ligand
for this protein based on the chemical properties of this ligand we have
chosen site no1 as the probable active site which comprises of ASP18
MET20 SER77 GLY130 TRP184 ALA87 LYS235 PRO87 etc The CD
domain predicted the protein to have GST-C-SUPERFAMILY which generally
takes part in cellular detoxification by catalyzing the conjugations (GSTs) with
a wide range of endogenous and xenobiotic alkylating agents The predicted
active site shows big cavity In that big cavity we docked two things one
cofactor Glutathione and other ligand DDT Glutathione was chosen because
we observed slight enhancement of enzyme activity with the addition of
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
305
glutathione Glutathione cofactor was docked first followed by DDT ligand
The potential energy of the overall reaction was -324278kcalmol
In general blast search of the enzyme showed sequence homology to
that of bacterial acetolactate synthase With specific blast search with the
reported sequence of DDT resistance gene of Aedes aegypti the sequence
showed great similarity with GST of Aedes aegypti However its identity was
observed to be only 59 Then second similarity was shown with GST of
Culex quinquefasciatus The blast search revealed that the sequence identity
between the VGSC of An gambiae and VGSC of Homo sapiens was 312
The DDT-dehydrohalogenase protein showed a molecular weight of
2778936 and isoelectric point of 921 Using protaparam the physical and
chemical properties of DDT-dehydrohalogenase protein was found out
Number of amino acids found was 238 with a formula is
C1234H1927N345O341S23 Total number of atoms is 3870
Radar tool showed no repeats in DDT dehydrohalogenase protein
sequence because protein sequence had no repeats Motif indicated
structural part of protein Number of Motif found in DDT dehydrohalogenase
was 125 and block showing an ungapped aligned motif consisting of
sequence segments that are clustered to reduce multiple contribution from
groups of highly similar or identical sequences Here CD domain predicted
that glutathione-s-transferase is the domain of DDT dehydrohalogenase
protein Its e-value is 7e-10 Prosite tool showed no pattern and profile in
DDT dehydrohalogenase protein sequence because the protein sequence
doesnrsquot contain pattern and profile The average residue weight was 116762
and charge was 238 Pepinfo tool showed the plot of hydropathy value and
hydropathy plot of residues There were large number hydrophobic
aminoacids Most hydrophobic aminoacids of DDT dehydrohalogense are
isoleucine(45) and valine(42) The most hydrophyilic one argenine (-45)
and lysine This is very important in protein structure Hydrophobic
aminoacids tend to be internal while hydrophilic amino acids are more
commonly found towards the protein sequence Pep window tool shows kyte-
doolittle plot and hydropathy It also shows Tiny residue (A+C+G+S+T)
small residue(A+B+C+D+G+N+P+S+T+V aliphatic residues (I+L+V)
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
306
aromatic residues (F+H+W+Y) non-polar residues
(A+C+F+G+I+L+M+P+V+W+Y) polar residues (D+E+H+K+N+Q+R+S+T+Z
charged residues (B+D+E+H+K+R+Z) in the protein DDT
dehydrohalogenase The enzymes such as Asp-n-endopeptidase Asp-n-
Endopeptidase+n-terminal glu BNPS-skatole CNBR Chymotrypsin high-
specificity chymotrypsin etc cleaved the protein DDT dehydrohalogenase
sequences
Secondary structure prediction of FAP52 based on its primary
structure indicated that itrsquos NH2-terminus has a long stretch with an -helical
arrangement with a high-degree of propensity to coiled-coil arrangements
This could be fulfilled either by intra- or inter molecular interactions In order
to distinguish between these possibilities the capacity of FAP52 to self-
associate were analysed The PAIRCOIL program predicted that FAP52
included three regions comprising the residues 146ndash179 185ndash219 and 248ndash
280 with the probability of 53 100 and 9 respectively to form coiled-
coils While another program MULTICOIL further discriminated the
propensity for dimeric and higher oligomeric arrangements giving the
likelihoods of dimer formation of 3 74 and 0 and of trimer formation of
4 10 and 05 for the same regions respectively Here the PAIRCOIL
program predicted that DDT dehyrohalogenase had no coiled coils in the
residue position and MULTICOIL predicted that the probability cut off for the
coiled-coil locater was 050 The scoring distances for dimeric table was 2 3
4 for trimeric table it was 1 2 3
For construction of 3D model of VGSC protein of An gambiae the
retrieved sequence was submitted to PSI-BLAST The comparative model
failed to find a suitable template for the VGSC sequence Therefore
threading method was employed to find an appropriate template using 3D
Position Specific Scorings Matrix server (3DPSSM)
(httpwwwsbgbioicacuk~3dpssm) The server predicted voltage-gated
potassium channel (VGPC) of A pernix as a template for the domain II of
VGSC For the other regions of VGSC no template with the required
sequence identity of gt20 was found Therefore a 3D model was
constructed only for domain II of VGSC using MODELLER programmes
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
307
httpsalilaborgmodellerdownload_installationhtml) For construction of 3d
model of DDT-dehydrohalogenase of Pseudomonas putida T5 the retrieved
sequence was submitted to PSI-BLAST The comparative model failed to find
a suitable template for the DDT-dehalogenase sequence Therefore threading
method was employed to find an appropriate template using 3DPSSM or
PHYRE online software Many structures were predicted by phyre software
Among these structures one structure was selected based on its sequence
identity and e value
Ramachandran plot of VGSC Domain II of each of the 20 amino
acids that included glycine and proline the ADIT validation indicated that the
VGSC model had 6 amino acids in unfavourable region where as in the
template the unfavourable region contained a total of 19 amino acids Thus
the analysis (in the absence or in the presence of glycine and proline) clearly
suggested that the backbone conformations of the model have suitable
structural quality when compared with the template Ramachandran plot of
Pseudomonas putida T5 of each 20 aminoacids that included glycine proline
and aparagine (the ADIT validation) indicated that the asparagine or
aspartate in the side chain formed a hydrogen bond with the main chain and
therefore stabilised the enzyme which was otherwise an unfavourable
conformation Disallowed regions generally involved steric hindrance between
the side chain C-beta methylene group and main chain atoms The active
sites of the VGSC domain II model were predicted by the PASS programme
In the modelled protein the ASTp program illustrated the presence of a total
of 30 pockets meant for ligands interaction Here the active site of DDT-
dehydrohalogenase of Psueodomonas putida T5 was predicted by binding
site molecule of discovery studio 20 docking software which predicted 6
possible active sites Based on the chemical properties of the ligand site no1
was chosen as the probable active site which comprised of SP18 MET20
SER21 CYS42 TRP 129 AG117
The side chain conformations of the constructed model of
Pseuedomonas putida T5 domain were refined to minimize the error DDT
dehydrochlorinase docking result had been reported previously in A gambaie
GSTs but this is the first report of docking DDT-dehydrohalogenase in
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
308
Pseudomonas putida T5 Here the predicted domain is GST_C family Class
Theta subfamily composed of eukaryotic class Theta GSTs and bacterial
dichloromethane (DCM) dehalogenase GSTs are cytosolic dimeric proteins
involved in cellular detoxification by catalyzing the conjugation of glutathione
(GSH) with a wide range of endogenous and xenobiotic alkylating agents
including carcinogens therapeutic drugs environmental toxins and products
of oxidative stress and here the cofactor glutathione is defined as tripeptide
with many roles in cells It conjugates to drugs to make them more soluble for
excretion is a cofactor for some enzymes is involved in protein disulfide
bond rearrangement and reduces peroxides
The report of A gambiae GSTs are of particular interest because of
their involvement in resistance to DDT [1 1 1 -trichloro-22-bis-(p-
chlorophenyl) ethane] which is an important malaria vector In A gambiae
an increased rate of DDT dehydrochlorination in the resistant strain was
associated with quantitative increase in multiple GST enzymes The report of
Aedes aegypti found no evidence for increased levels of this GST protein in
DDTpyrethroid-resistant populations from Thailand In A gambiaea also
gluthathione was cofactor Glutathione in Drosophilla indicated detoxification
and is of interest for two reasons First the ecological niche of Drosophila
species is largely defined by the larval feeding substrate and therefore the
genes enabling Drosophila to identify detoxify and utilize these substrates as
nutritional resources are obvious candidates for genes that have been the
targets of natural selection Glutathione S-transferases encoded in the
D melanogaster genome are divided into two nonhomologous families the
microsomal GSTs for which there are three genes and the canonical GSTs
that are cytosolic (36 genes)
Docking result of VGC DOMAIN was done with this altered model
the ligand DDT interacting with a different group of residues such as glutamic
acid methionine cysteine leucine tyrosine and phenylalanine with a binding
energy of minus621 kcalmol Here docking result of Pseudomonas putida T5 was
done with ligand DDT with a differing group of residues such as arg lys leu
tyr leu with a binding energy is -324278 kcalmol DDT was docked within
predicted catalytic sites by using the DOCK function of MOE in case of
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
309
Agambaie Here DDT was docked within the predicted active sites by using
DOCK function of MOE of Pseudomonas putida T5 It is taken as a ligand In
Pseudomonas putida T5 and Agambaie Visual inspection indicated that
DDT dock best in the presence of glutathione with the trichloro group of DDT
orientied toward the sulphur atom of glutathione This conformation agreed
that with GSH depended dehydrochlorinase activity proposed by Matsumura
(1985) In the Anopheles gambiae the side chains of CYP6Z1 are predicted
to be distant enough from DDT to be free of van der Waals clashes But in
Pseudomonas putida T5 the vander waals radii were completely attached with
DDT
4A5 Conclusion
DDT- dehydrohalogenase is an enzyme that is involved in the removal
of one chlorine from DDT molecule The gene responsible was isolated from
Pseudomonas putida T5 by PCR cloned and expressed in E coli BL 21
using pET 28a plasmid vector
In the computational study the enzyme sequence deciphered by gene
sequence was used for the construction of models employing homology
modelling method Molecular docking of DDT ndashdehydrohalogenase with DDT
substrate was subsequently studied Docking was also done along with GSH
a co-factor like substrate These two were found to have different binding
pockets regarding the size and the key amino acids involved in binding
Predicted binding modes of these two with DDT ndash degrading enzyme was
compared The calculated docking interaction energy of DDT showed high
affinity suggesting specificity of the enzyme
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
310
References
Alexey G Murzin Steven E Brenner Tim Hubbard Cyrus Chothia 1995
SCOP A structural classification of proteins database for the
investigation of sequences and structures Journal of Molecular
Biology 247(4)536ndash540
Barker PS Morrison FO 1964 Breakdown of DDT to DDD in mouse
tissues Canadian J Zool 42324ndash325
Bennett-Lovsey RM Herbert AD Sternberg MJ amp Kelley LA 2008 Exploring
the extremes of sequencestructure space with ensemble fold
recognition in the program Phyre Proteins 70 611-625
Benning MM Wesenberg G Liu R Taylor KL Dunaway- Mariano D Holden
HM 1998 The three-dimensional structure of 4- hydroxybenzoyl- Co A
thioesterase from Pseudomonas spp strain CBS- 3 Journal of
Biological Chemistry 273 33572- 33579
ChengJ Randall AZ Sweredoski MJ Baldi P 2005 SCRATCH a protein
structure and structural feature prediction server Nucleic Acids Res
33 web server issue W72ndashW76
Cuff J A and Barton G J 1999 Evaluation and Improvement of Multiple
Sequence Methods for Protein Secondary Structure
Prediction PROTEINS Structure Function and Genetics 34508-519
de Jong RM Dijkstra BW 2003 Structure and mechanism of bacterial
dehalogenases different ways to cleave a carbon-halogen bond
Current Opinions in Structural Biology 13 722- 730
de Jong RM Tiesinga JJ Rozeboom HJ Kalk KH Tang L Janssen DB
Dijkstra BW 2003 Structure and mechanism of a bacterial haloalcohol
dehalogenase a new variation of the short-chain dehydrogenase
reductase fold without an NAD (P) H binding site European Molecular
Biology Journal 22 4933- 4944
Hisano T Hata Y Fujii T Liu JQ Kurihara T Esaki N Soda K 1996 Crystal
structure of L-2-haloacid dehalogenase from Pseudomonas spp YL
An alpha beta hydrolase structure that is different from the alpha beta
hydrolase fold Journal of Biological Chemistry 271 20322- 20330
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
311
Jones DT 1999 Protein secondary structure prediction based on position-
specific scoring matrices J Mol Biol 292(2)195-202
Kallman BJ Andrews AK 1963 Reductive dechlorination of DDT to DDD by
yeast Science 141(3585)1050-1
Kelley LA and Sternberg MJE 2009 Protein structure prediction on the web
a case study using the Phyre server Nature Protocols 4 363 ndash 371
Mannervik B Board PG Hayes JD Listowsky I Pearson WR 2005
Nomenclature for mammalian soluble glutathione transferases
Methods Enzymol 4011-8
Marchler-Bauer A S Lu J B Anderson et al 2011 CDD a Conserved
Domain Database for the functional annotation of proteins Nucleic
Acids Res 39(D) 225-9
Matsumura F 1985 Toxicology of Insecticides Plenum Press New
York
McGuffin LJ Street S Soslashren-Aksel Soslashrensen and David T Jones 2004 The
Genomic Threading Database Bioinformatics applications note
20(1)131ndash132 (DOI 101093bioinformaticsbtg387)
Nagata Y Mori K Takagi M Murzin AG Damborsky J 2001 Identification of
protein fold and catalytic residues of gamma- hexachlorocyclohexane
dehydrochlorinase LinA Proteins 45 471- 477
Poelarends GJ Zandstr M Bosma T Kulakov A Larkin MJ Marchesi JR
2000b Haloalkane utilizing Rhodococcus Strains isolated from
geographically distinct locations possess a highly conserved gene
cluster encoding haloalkane catabolism Journal of Bacteriology 182
2725- 2731
Ridder IS Rozeboom HJ Dijkstra BW 1999 Haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 refined at 115 Aring resolution
Acta Cryst D551273 ndash 1290
Seffernick JL de Souza ML Sadowsky MJ Wackett LP 2001 Melamine
deaminase and atrazine chlorohydrolase 98 percent identical but
functionally different Journal of Bacteriology 183 2405- 2410
Supariya S 2009 prediction of molecular structures of DDT
dehydrohalogenase and docking
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014
Chapter 2
312
Trantirek L Hynkova K Nagata Y Murzin A Ansorgova A Sklenar V
Damborsky J 2001 Reaction mechanism and stereochemistry of
gamma- hexachlorocyclohexane dehydrochlorinase LinA Journal of
Biological Chemistry 276 7734- 7740
Ward JJ McGuffin LJ Bryson K Buxton BF and Jones DT 2004
The DISOPRED server for the prediction of protein disorder
Bioinformatics applications note 20(13) 2138ndash2139
(doi101093bioinformaticsbth195)
Wedemeyer G 1967 Dechlorination of 111-Trichloro-22-bis(p-
chlorophenyl) ethane by Aerobacter aerogenes Applied Microbiology
15569- 574
Zheng J Cho M Jones AD and Hammock BD 1997 Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of murine Clara cells after exposure to naphthalene Chem
Res Toxicol 10 1008 ndash1014