1. Biochemical and Biophysical Characterization of AnAEst, a
novel SGNH hydrolase Kiranmayee Bakshy Indian Institute of
Technology Madras, Chennai, India PI: Dr. Manoj Narayanan 1
2. Contents Comparative analysis (structural and functional
evolution) Biochemical characterization (functional and kinetic
analysis) Biophysical characterization (thermal stability structure
function relationship) 2
3. Tools set Sequence and structural homology searches DALI,
HHPRED, PDB Sequence and structural alignment tools FATCAT,
TCOFFEE, MULTIPROT Molecular visualization tools PyMol, RasMol,
SwissPDB Protein over expression in E.coli BL21, rosetta strains
Protein purification using Ni-NTA column chromatography followed by
size exclusion chromatography Site-directed mutagenesis
(Stratagene) for active site mutants generation Enzyme activity
assay methods: titrimetry, HPLC, colorimetry, spectrophotometry,
fluorimetry, zymography, TLC Biophysical techniques: DSC, far and
near UV CD, fluorescence spectroscopy and Trp fluorescence
quenching studies, ANS binding studies, fourth derivative spectra
Crystallization: hanging drop method; using Hampton crystallization
screens Ligands used for co-crystallization: AEBSF, PMSF,
imidazole, acetate 3
4. 4 Serine hydrolases Structural classification of Serine
hydrolases Beta proteins Alpha/beta proteins Trypsin-like serine
proteases Crotonase-like (Seq-10821; Str-59) Methylesterase
C-domain (Seq-1586; Str-2) Subtilisin-like (Seq-6532; Str-160) /
Hydrolase (Seq-28102; Str-750) Flavodoxin-like (SGNH hydrolases)
Gariev, IA. and Varfolomeev, SD. (2006) Bioinformatics 22,
2574-2576 (Seq-5119; Str-13) In 1995, Upton and Buckley identified
new class of lipolytic enzymes In 2003, this class has been named
GDSL group of serine lipases/esterases SGNH hydrolases are widely
spread across all taxa
5. 5 / hydrolase fold SGNH hydrolase fold Structural comparison
P. aeroginosa lipase PDB ID 1EX9 PDB ID 1IVN E. coli TAP Compact
fold performs multiple functions Biochemical studies available for
very few of them
6. 6 Flavodoxin fold substantially different from the canonical
/ hydrolase fold N C 3 1 2 45 1 2 345 6 7 NuAc H Topological
differences SGNH hydrolase fold E. coli TAP Canonical / hydrolase
fold P. aeroginosa lipase N C 1 2345 6 123 45678 NuAc H -helix
-strand Blue colour - insertions with respect to SGNH hydrolase
fold Mala and Takeuchi, Anal Chem Insights (2008),3, 919 Akoh,CC et
al., Progress in Lipid Res. (2004), 43, 534552
7. Structure based sequence alignment 7 / hydrolases SGNH
hydrolases SGNH family members can be identified only from these
four blocks
10. 10 Structure based sequence alignment of SGNH hydrolases
SGNH hydrolases share a very low sequence identity Catalytic
residues are structurally conserved
11. 11 RGAE-TAP MsAct-TAP AnAEst-TAP Bt12063b-TAP Loop 1 Loop 2
Tertiary structural variations around the active site cleft can be
implicated to diverse substrate specificity Structural basis for
diversity in substrate specificity
12. 12 Highly conserved tertiary structures and catalytic site
Well conserved tertiary structures in spite of the presence of
highly variant primary structure TAP SsEst Active site rmsd ranges
from 1.5-3.2
13. 13 Structural basis for diversity in quaternary structure
The diversity in oligomerization and substrate specificity can be
attributed to specific secondary structural insertions Side-by-side
(II type) dimer Back-to-back (X3 type) dimer -helix -strand Blue
colour - insertions with respect to E.coli TAP
14. Conclusions Flavodoxin fold is substantially different from
the canonical / hydrolase fold - hence the name SGNH hydrolase fold
SGNH family members can be identified only from the four conserved
sequence blocks SGNH hydrolases share a very low sequence identity
and the catalytic residues are structurally well conserved Tertiary
structures are well conserved in spite of the presence of highly
variant primary and quaternary structure The diversity in
oligomerization and substrate specificity can be attributed to
specific secondary structural insertions 14
15. 15 Expression, purification and biochemical
characterization of AnAEst
17. Regular biochemical characterization AnAEst is an
arylesterase which hydrolyses small chain fatty acid aryl esters It
exhibits an optimal activity at pH 7.5 and in a broad temperature
range 25-45 C Among all the divalent cations Cu+2 and Fe+2 shows
inhibitory effect of the esterase activity What are the active site
residues to be considered for mutational and kinetic studies ? 17
Bakshy K, Gummadi SN, Manoj N, Biochim Biophys Acta. 2009,
2:324-334
18. 18 Selection, generation and purification of active site
mutants L86 R54 S17 PDB ID 1z8h WT S17A R54G L86A M kDa 80 66 56 40
29 25 20 17 14 SDS-PAGE analysis of purified AnAEst and its mutants
Wild-type and mutants were purified under similar conditions
Selection of AnAEst mutations The following residues were selected
and mutated by site-directed mutagenesis: S17 nucleophile Ala R54
oxyanion Gly L86 active site Ala
19. 19 WT S17A R54G L86A Zymogram showing the activities of
AnAEst and its mutants Arylesterase zymogram : 1-NA, Fast blue B
Native PAGE for basic proteins under neutral conditions Altered
specific activity of R54G mutant with increased activity against
phenyl esters Standard assay condition: 50 mM sodium phosphate (pH
7.5); 1 mM substrate; 2 g purified enzyme; at 25 C. Results
displayed are mean of three individual experiments Determination of
activity profile of active site mutants Spectrophotometric assays
using various substrates Bakshy K, Gummadi SN, Manoj N, Biochim
Biophys Acta. 2009, 2:324-334.
20. 20 Substrate Kinetic parameters Wild-type R54G L86A
-naphthyl acetate Km (mM) kcat (x103min-1) kcat /Km (x103mM-1min-1)
0.280.01 1.32 4.71 0.610.02 0.96 1.61 0.280.05 0.18 0.64 -naphthyl
propionate Km (mM) kcat (x103min-1) kcat /Km (x103mM-1min-1)
0.710.05 0.36 0.51 2.060.67 0.36 0.17 0.240.02 0.05 0.21
p-nitrophenyl acetate Km (mM) kcat (x103min-1) kcat /Km
(x103mM-1min-1) 2.440.31 6.36 2.60 6.350.50 26.50 4.17 3.700.46
1.44 0.39 Phenyl thioacetate Km (mM) kcat (x103min-1) kcat /Km
(x103mM-1min-1) 3.300.42 6.14 1.86 6.460.51 29.30 4.53 2.140.16
1.35 0.63 Results displayed are mean of three individual
experiments Standard assay condition: 50 mM phosphate pH 7.5;
varied [substrate]; 2 g purified enzyme; at 25 C. Kinetic
parameters of AnAEst and its active site mutants Wild-type shows
highest affinity and catalytic efficiency to 1-NA R54G shows
highest affinity to 1-NA whereas highest catalytic efficiency to
PTA L86A shows highest affinity to 1-NP whereas highest catalytic
efficiency to 1-NA
21. 21 Enzyme Accessible surface area (2) Cavity volume (3)
Cavity length () WT R54G L86A 21.9 21.9 32.2 3.3 3.3 8.8 27.5 27.5
38.8 Active site dimensions of AnAEst and its mutants Rationale for
different substrate specificities of mutants Different binding
modes of phenyl and naphthyl esters Location of R54 and salt bridge
formation with E92 Conversion of Michaelis complex to tetrahedral
complex could involve movement of amide protons of R54 during
oxyanion formation PDB ID 1z8h L86 R54 S17 E92 1NA Y128 D179 H182
F18 N87 R54 and L86 are important in substrate binding and
catalysis Bakshy K, Gummadi SN, Manoj N, Biochim Biophys Acta.
2009, 2:324-334.
22. Biophysical characterization of AnAEst pH and thermal
stability 22
23. 23 Results displayed are mean of three individual
experiments Thermal deactivation of AnAEst Process of deactivation
is irreversible The enzyme follows first order deactivation
kinetics Enzyme was incubated for different time periods at
different combinations of pH and temperature whose residual
activity was measured at standard assay conditions Standard assay
condition: 50 mM sodium phosphate (pH 7.5); substrate-0.6mM 1-NA; 2
g purified enzyme; at 25 C. tk t d eEE )( 0 DE dk
24. 24 Low pH, low temp. - 80-100 % residual activity High pH,
low temp. 25-75 % residual activity At 50 C, as pH increases
residual activity decreases from 75-25 % At 60 C, 2 % activity
remains at all pH within 2 hrs The lines are fitted to first order
deactivation kinetic equation with R2>0.9 Thermal deactivation
of AnAEst
25. 25 The deactivation rate constant (kd) can be obtained from
the slope of the plot ln(Et/E0) vs Time Half-life was calculated
from the Eq. below: Half-life of the enzyme decreases with increase
in pH and temperature. Maximum half-life was observed at 30 C and
pH 5.5 indicating its maximum stability at these conditions Optimum
conditions of activity and stability for AnAEst are different
Optimum activity conditions : pH 7.5, 25-45 C Optimum stability
conditions : pH 5.5, 25-45 C dk t 693.0 2/1 Thermal deactivation of
AnAEst
26. 26 DSC was performed to monitor the structural stability or
thermal unfolding of AnAEst, but the protein tends to aggregate
beyond 70 C Transition peak, Tp at pH 5.5 and 7.5 are 64.5 and 60.2
C respectively Effect of pH on Molar heat capacity of wild-type
AnAEst
27. 27 Structural stability : CD spectra The residual secondary
structures correspond to the residual activity of the protein Near
UV-CD spectra showed presence of tertiary structures at all
conditions Complete loss in secondary structures was not observed
so what is happening to the microenvironment of the aromatic
residues ? pH 5.5
28. 28 Decrease in intrinsic Trp fluorescence along with a red
shift indicates exposure of Trp to polar solvent Structural
stability : Trp emission spectra Enzyme was incubated for different
time periods at different combinations of pH and temperature whose
residual fluorescence was measured at pH 7.5 and 25 C with
excitation wavelength of 290 nm
29. 29 Structural stability : fourth derivative spectra To
determine the microenvironment of other aromatic residues such as
Tyr and Phe UV absorption spectra of the incubated protein was
recorded which was converted to 4th derivative spectra Peak at 260
nm Phe 275 nm Tyr 292 nm - Trp Decrease in peak intensity was
observed with increase in temperature at 260 nm, 275 nm and 292 nm
Microenvironment of the aromatic residues becomes more polar This
indicates opening up of the enzyme structure
30. 30 Protein dynamics-Tryptophan quenching Slope of the plot
F0/F vs quencher concentration gives Ksv, Stern-Volmer constant
Linear plots static/dynamic quenching; positive deviation from
linearity static and dynamic quenching Modified Stern-Volmer
equation for positive deviation from linearity: QK F F sv10 0 1 [
]app F K Q F 0 1 1 [ ] app D S D S F K K K K K Q F Q
31. 31 Protein dynamics-Tryptophan quenching Enzyme was
incubated for 1hr at different combinations of pH and temperature
and titrated with the quencher at pH 7.5 and 25 C. * Indicates Kapp
or Ksv Acrylamide Ksv at all temperatures for pH 5.5 > 7.5 and
9.5 states indicating higher diffusion of acrylamide through the
protein matrix At pH 5.5, Ksv remains constant with increase in
deactivation temperature indicating nearly same extent of quenching
Fluorescence studies indicate that the enzyme states incubated at
pH 5.5 is blue shifted (~2-4 nm) in comparison with those incubated
at pH 9.5 indicating buried Trp
32. 32 KI Protein dynamics-Tryptophan quenching Enzyme was
incubated for 1hr at different combinations of pH and temperature
and titrated with the quencher at pH 7.5 and 25 C. * Indicates Kapp
or Ksv Larger Ksv values at all temperatures observed for pH 5.5
> 7.5 and 9.5 Varying Ksv trends observed for different pH
states At pH 5.5, Ksv remained constant with increase in
deactivation temperature but with a sharp increase at 60 C This
varying behavior of quenching by KI at different pH states can be
attributed to the varying charge around the microenvironment of Trp
residues Electrostatic interactions seem to play a crucial role in
determining the structural stability of AnAEst What happens to the
hydrophobic regions of the protein ?
33. 33 Structural stability : ANS binding spectra With increase
in temperature, ANS binding increases indicating increased exposure
of hydrophobic regions on the protein At 60 C, ANS binding
decreases with increase in pH Maximum hydrophobic patches can be
observed at pH 5.5 and 60 C 60 C Size exclusion analysis of AnAEst
after incubating at pH 5.5, 7.5 and 9.5 separately at 45 and 60 C
revealed that the protein exists as a dimer This indicates that the
protein exhibits a high degree of conformational plasticity in its
core dimeric structure
34. Conclusions Enzyme is stable at pH 5.5 from 25-45 C, for
6-8 hrs and follows a first order deactivation kinetics Thermal
deactivation occurs as a result of protein unfolding gradually
exposing the hydrophobic regions of the protein The highest thermal
stability of AnAEst exposed to pH 5.5 is mostly due to the global
conformational changes involving unique ionic interactions 34
35. 35 Crystallization of Wt-AnAEst Crystals were observed in
0.1 M MOPS pH 6.8, 11 % (w/v) PEG 4000 and isopropanol 9 & 10 %
(v/v) at 21 C. Crystal fine screens were set up to reproduce the
previously formed crystals of AnAEst. Various ratios of reservoir
solution: protein was also used (1:1, 1:2, 2:1) at the above
mentioned conditions. Crystals were observed in the fine screens
after about 3 months at almost the same conditions. 0.1 M MOPS pH
6.6, 11 % (w/v) PEG 4000 and isopropanol 9, 11 and 13 % (v/v) at 21
C 1) 0.1 M HEPES sodium pH 6.8, 10% (v/v) isopropanol, 11% (w/v)
PEG 4000, 4C, 25 mg/ml protein conc. 2) 0.1 M MOPS pH 6.8, 11 %
(w/v) PEG 4000 and isopropanol 9 & 10 % (v/v) at 21 C
36. 36 Acknowledgements The Department of Science and
Technology, New Delhi, India. The Bioinformatics Infrastructure
Facility at IITMadras The Genomics Institute of the Novartis
Research Foundation, USA, for their kind gift of the clone of
AnAEst Department of Biotechnology, IITMadras HOD Prof K. B.
Ramachandran Prof G. K. Suraish Kumar Supervisor: Dr. Manoj
Narayanan Doctoral committee members Dr. G. Satyanarayana Naidu Dr.
A. Gopalakrishna Prof D. Loganathan Department of Chemistry,
IITMadras Prof A. K. Mishra - Department of Chemistry, IITMadras
Dr. V. Kesavan, Department of Biotechnology, IITMadras Prof K.
Suguna and group MBU, IISc, Bangalore Dr. R. Sankaranarayanan and
group CCMB, Hyderabad Prof Shekar C. Mande CDFD, Hyderabad Prof
M.J. Swamy and group Hyderabad University, Hyderabad Friends and
labmates: Sirisha, Navin, Ravi, Santosh, Harshavardhan, MJ,
Madhavi, Sai Krishna, Prashant, Prabhahar, Vidya, Vipin, Jayakumar,
Abhipsa, Shyam, Swati, Santosh, Aneesh and others Family
