Chapter4

Biochemical Characterization of FadD9 protein

Chapter4

4.1 INTRODUCTION

The FadD9 protein is the largest of 34 FadD paralogues in Mycobacterium

tuberculosis H37Rv (Cole et al., 1998). This protein showed high homology with

carboxylic acid reductase (CAR) protein from Nocardia sp. Strain NRRL 5646.

The CAR enzymes are relatively new and unexplored class of enzymes. The CAR

protein contained three domains: acyl-activating (A), acyl-carrier protein (ACP, T)

and reductase (R) domain. The best known example is a-aminoadipic acid

reductase (AAR, Lys2), a homologous protein involved in biosynthesis of lysine in

Saccharomyces cerevisiae. The adenylation (A) domain of AAR activates a­

aminoadipic acid with ATP as a-aminoadipoyl-AMP and transfers it to the

phosphopantatheine arm of ACP. The reductase (R) domain reduces P-pant bound

thioester substrate to aldehyde as a-aminoadipate-8-semialdehyde. The reductase

domains similar to AAR proteins have been characterized in non-ribosomal

peptide synthetase (NRPS) proteins. These domains are present as termination

domains and release the newly synthesized peptide as an aldehyde. The aldehyde

can undergo a variety of modification by accessory enzymes. For example, it could

be reductively transaminated to form a terminal amine as seen in the case of lysine

biosynthesis (Ehmann et al., 1999), condensed with an amino group to form

intramolecular Schiff base as seen in saframycin Mx1 from Myxococcus xanthus

(Pospiech et al., 1996; Pospiech et al., 1995). Another possibility involves

complete reduction of C-terminal residue to form an alcohol as observed in

peptaibols (Keating et al., 2001). Interestingly, the reductase domain in

myxochelin synthase can perform two rounds of iterative reduction to synthesize

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Chapter4

alcohols of newly synthesized non-ribosomal peptides (Gaitatzis et al., 2001;

Gaitatzis et al., 2005).

This mode ·Of aldehydes or alcohol synthesis is rather unique from the more

commonly employed mode of synthesis. Primarily, acyl-CoA reductase proteins

catalyze the reduction of fatty acyl-CoA thioesters to aldehydes and alcohols by

using one or two molecules ofNADH+W (Vioque and Kolattukudy, 1997; Wang

and Kolattukudy, 1995). Although, direct reduction of carboxylic acids has been

observed with microbial whole cell lysates, the enzymes responsible for these

reactions had stayed elusive. The Nocardia CAR protein was purified from cell

lysate and identified as the enzyme responsible for reduction of benzoic acid to

benzaldehyde by using one molecule each of ATP and NADPH (He et al., 2004; Li

and Rosazza, 2000).

In this chapter, we have demonstrated that FadD9 is a fatty acid reductase

and reduces fatty acids to corresponding aldehydes. We have biochemically

characterized the catalytic function of both adenylation and reductase domain. The

products formed by both the domains are characterized by a combination ofRadio­

TLC, analytical HPLC and mass spectrometry approaches.

4.2 METHODS

4.2.1 Characterization of FadD9 activity

For the characterization of acyl-adenylate forming domain, the typical

reactions were performed as described in chapter 2. Reactions contained 50mM

Tris buffer pH 7.5, 60J.!M 14C-lauric acid, 240,.M unlabelled lauric acid, 8mM

MgCh, 2mM ATP and 25pmol of purified protein. The reactions were quenched

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Chapter4

with 10% acetic acid after 15 minutes incubation at 30°C and analyzed by running

TLC plates.

For the reductase domain activity assays, reactions contained 50mM Tris

buffer pH 7.5, 60J.!M 14C-lauric acid, 240J.!M unlabelled lauric acid, 8mM MgCh,

2mM ATP, 5mM NADPH and 25pmol of purified protein. The reactions were

quenched with 10J..ll of 10% glacial acetic acid and extracted twice with 400J..tl of

ethylacetate. The extract was dried under vacuum and the residue was resuspended

in 20J..ll methanol. The products were resolved on silica gel 60 F254 TLC plates

(Merck) in 90% Hexanes in ethylacetate (v/v). The radioactive spots were

analyzed by phosphorimager.

Spectrophotometric assays were also performed to estimate the reductase

activity by following the NADPH utilization at 340nm wavelength. 500J..ll

reactions were set up with 1 OOpmoles of FadD9 protein containing reaction

components at above mentioned concentrations. The reference cuvette contained

all components except enzyme, and the background control cuvette contained all

components except benzoate. . The background control was subtracted from the

complete reaction to give the true enzyme activity. Reactions were initiated by

adding enzyme and were monitored at 340nm at 25°C. Rate of product formation

was calculated by decrease in NADPH absorption per min.

4.2.2 MS Identification of the A-Domain products

The FadD reaction products were loaded on C8-reverse phase columns

(Phenomenex). Ternary solvent system was employed to resolve various reaction

components from FadD products. 20mM ammonium acetate Buffer pH 5.4 was

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Chapter4

used as solvent A and methanol/acetonitrile, 15/85 (v/v) as solvent B. The elution

profile was monitored at 254nm. Reaction products were analyzed using

nanospray ESI-MS (API QSTAR Pulsar i MS/MS, Applied Biosystems).

4.2.3 Transfer of activated Carboxylic acids to ACP domain

FadD9 protein was incubated along with 2mM ATP, 8mM MgCh and

100~ 14C-fatty acid at 30°C for 15min. Reactions were quenched with SDS gel

loading dye and the proteins were separated using SDS-PAGE gel. The gels were

stained with Coomassie Brilliant Blue R250 and soaked in Amplify for 15min.

These were dried and exposed to phosphorimager. In the control reactions, ATP

was excluded from the reaction mixture.

4.2.4 MS ldentificati9n of the R-Domain products

The reaction products were resuspended in 50J.Ll methanol and loaded on

C8-reverse phase columns (Phenomenex). !socratic gradient of methanol

containing 10% dichloromethane was employed to resolve various reaction

products. The elution profile was monitored at 214nm. Reaction products were

analyzed using nanospray ESI-MS (API QSTAR Pulsar i MS/MS, Applied

Biosystems).

4.2.5 MS identification of the aldehyde adducts

Aldehydes are known to be reactive intermediates and make adducts with

free amino groups present on proteins and other metabolites. The

benzyloxycarbonyllysine methyl ester (z-Lys-OMe) was used as a peptide mimic.

The unstable Schiff base adduct was reduced with NaCNBH3, a mild reducing

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agent. The a-amino-alkyl-z-Lys-OMe could be analyzed by mass spectrometry.

The identities of compounds were confirmed by using nanospray ESI-MS (API

QSTAR Pulsar i MS/MS, Applied Biosystems).

4.3 RESULTS

4.3.1 Computational analysis of the FadD9 protein

To investigate whether FadD9 is conserved across mycobacterial species,

we analyzed various mycobacterial genomes by Blast. The FadD9 paralogues

could be identified in several mycobacterial species. The FadD9 protein from M

tuberculosis H37Rv, M tuberculosis CDC1551 and M bovis AF2122/97 were

more than 90% identical. The FadD9 also showed high sequence identity with

carboxylic acid reductase proteins from Nocardia sp. NRRL 5646 and Nocardia

farcinica IFM 10152. The phylogenetic tree generated by multiple sequence

alignment of these proteins is shown in figure 4.1. This analysis suggests that

FadD9 is a evolutionary conserved protein and might be ~~mportant for

mycobacterial survival.

The FadD9 protein is the largest FadD protein in Mycobacterium

tuberculosis with the 1168 amino acid residues. The sequence analysis of FadD9

was performed using the in-house developed PKS-NRPS sequence analysis tool

(Ansari et al., 2004), that predicts NRPS domains and domain boundaries. This

analysis identified adenylation (A) and ACP (T) domains in FadD9 protein (Figure

4.2). TheN-terminal and A-T linker were predicted to be similar in size to typical

NRPS linkers. The C-terminal linker was 449 amino acid residues in length. This

size is rather big for C-terminal domain and we hypothesised that this could

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Chapter4

correspond to a domain not detected by PKS-NRPS-DB. The C-terminal linker

was therefore used for homology searches using Blast.

gl_ 1478222_FADD9_M_tb_H37Rv

,-----------------{[ gi_31793774_FADD9_M_bovis_A

gi_ 13882413_Mtb_Mtb_CDC1661

1

,---------- gi_41407138_M_Av_Paratb

'------------- gi_3136006_M_Ieprae

gi_92911894_M_sp._JLS

gi_1 08799238_M_sp._MCS

'------------- MSMEG2968_M smeg

'--------------gi_90206636_M_vanbaalenii_PY

,---------------gi_40796035_CAR_N_sp._NRRL_6

'------------,,---------- gi_64016491_CAR_N_farclnlca

[L_ ____________________ MSMEG6713 M smeg

Figure 4.1: The dendrogram obtained by multiple sequence alignment of proteins homologous to FadD9 protein using ClustalW.

Table 4.1: The details FadD9 paralogues identified in mycobacterial and nocardia species. The details for NRPS R domains LtxA and MxaA are also given in this table.

gi number Protein name organism

1478222 FadD9 Mycobacterium tuberculosis H37Rv

31793774 FadD9 Mycobacterium bovis AF2122/97

13882413 substrate-CoA ligase Mycobacterium tuberculosis CDC1551

81253426 Long-chain acyl-CoA synthetases Mycobacterium tuberculosis C

Mycobacterium avium subsp. 41407138 FadD9

paratuberculosis K-10

3136006 putative Acyl-CoA synthetase Mycobacterium /eprae

92911894 Thioester reductase Mycobacterium sp. JLS

108799238 Thioester reductase Mycobacterium sp. MCS

90205636 Thioester reductase Mycobacterium vanbaalenii PYR-1

40796035 A TP/NADPH-dependent carboxylic

Nocardia sp. NRRL 5646 acid reductase

54015491 putative carboxylic acid reductase Nocardiafarcinica IFM 10152

76809770 LtxA Lyngbya majuscula

14210837 MxaA Stigmatella aurantiaca

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Chapter4

NRPS-PKS. D:\ T:\B:\SE OF ::\0"\"RIBOSO:\L\L PEPTIDE S\ ~THET.-\SES ~----

NRPS ACTINOMYCIN

ACV

BACILLIBACTIN

BACrnACIN

CDA

COMPLESTATIN

CYCLOSPORIN

EN'IlltOBACTIN

FENGYCIN

GRAMICIDIN

HCTOXIN

UCHENYCIN

SURF ACTIN

SYRINGOMYCIN

Fad09 Reductase

•click the domain images forfwtherdetails / Domain

~--·~

v

Figure 4.2: The results of PKS-NRPS sequence analysis display the predicted adenylation and ACP domains of FadD9 proteins.

As can be seen in the Figure 4.3(a), Blast analysis identified several

proteins and these proteins are presented in the Blast tree view. In general, three

families of proteins were detected to contain reductase (R) domains at C-terminus:

R domains present in NRPS modules, R domains present in nocardia CAR protein

and a-amino adipic acid reductase (AAR, lys2) homologues. As seen in Blast tree

view, the a-amino adipic reductase paralogues clustered as a separate group. This

may be explained by the fact that Lys2 proteins are monophyletic and are closely

related to each other (An et al., 2003).

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Chapter4

r--:=~~================~;;~~~~~~~~~=~opol!l«fideS~nthantchrom.. Peptidt s,...thetlst (Ba.dllu51huriMiensis :lltrO-omodulirpoi!A<tfide s~n1h,., rstreptomyces~~~m~~~itill$ m,.,.+seOJ

----0 ')-AT P-"!ADPH-de~dent cBJ'boHWc iWd reductase.

I putative Glbo~oeiAic ~reductase [Nocardia Wtinicalfhl peptide S}flfhebtst [Mycobaderium sme9m~:tisl non-ribosomal pep1idt ~thetase [Miol=obacterium smegmafi

peptide S!ofl1httase1 putative [~obamrium tuberculosis CD .. ,-------------~Ou PROB~BLE PEPTIDE SVNTHET~SENRP(PEPTIDE SVN.

COG1020: Non-ribo:somil peptide S}flthtfut modutts IJ'Id n ! -co~t020: Non-ribosom.i peptide SW~thetase modules and n ~PstB[Mycobacttriuma&.~iuml

"-----e PstA (M}.Ieobacitrillm avium subsp. ptnJuberculosis; ,;._

Uri eo&11ln Long. chain ""-CO~ s~nthttases (AMP.fonning) [Ml<O-<!l'PAOBABLE FATTY.ACIDCoA LIGASE FADD9 (F'ATT'Y-ACfO.COA.]

1:ub~_;~~~i9~e~~~e [M}Cobacferium tuberculosis CDC155t llm1~~~~ua.~ PI'On:ln PJ'Ou~M.I,-....

LJ PROBABLE FATTY. ACID-COlo. LIGASE F'AD09(FATTY.ACID-CoA~

.a Thioesternductase [bl}leoba.:1trium vanbaalenii P'YA-1) 0::::: ,-----<<ll:Thioesterreductase [Mycobocterium sp. JLS] Oct w, '<>ThioemmductaseiMI<obicttriumsp.MCSJ 1 r--0 F>dD9 [Ml'Cobamri11m ..,lum subsp. pan.tuben:ulosis K .. '--If' ~

1pol1; B1177_f2...70 (lrllo!Coba.cttrium lepruJ . put..fi'" ""-co~ s~nthttase [Ml'Coba<teri11m ..

put,.[We ~c"- co~ s~nthttase[llll<obamri11m .

L---[====~~~~~~~~~~~~~~~po~I!A<~e~fi~de~s~~n~epfideS~n1het...[Ml91lN>Ortht9rist~ h!oOOthefictJ proWn CHGG_OS266 [ChMtomlum globosum c". poJIA«fide s~nihue!Uibbmlllmonilifonni<J

poiJ.tetidt S!ol'lfhase tGibbertlti.momlifonnifl non-ribosomal peptide S~K~ihase [h1)M()coccus Hat'lihus OK 1622;1

MHcG (5figmattlli. iiWrirrfiaci4 non-ribosomaJ peptide S!Kithase triKCO (h1~coccos Kan1hus DK 1622J

non- ribosomal p:eptide s!Kihiase [Sfrtpfolm,lces auermi11lis tJtA. +680J "----------------<~Non-ribosomal peptide S!Kithtfise modults md rtlil.ttd protein (tiihella.

~=~~~~~~~~~~~~~~~~~~p~u-~·~e~llo~e~l p~o~I!A<~e~tide Sion1tw:t(SfrtpfomycescoeUcolorA:.(2D P'rla.tivt non- rlbosomt.l peptide S}.nthem:e (SfrtQtomyces rimosus subsp. pmmrom~o~t .•

h>oothtfic~ protein PWI' _o 1001183 [Pseudomonos wu9L peptide S!KithtDse (Agrobt.ettrium iume1i.::lens str. c 581

puh.five rtducb.H [Shpfomyces cotltcolor A3(2D • G~otht:ticafproteinurtro3;108.1 tusfilio9om~is 5211

~ ~ tlwotheiiuJ prottin MGOS290.<to [Ma~m~orthe grisea70.15J

'---------....:W ~ ~~:O~~~e:!~~~~;.\o~=~~z:en~i:;ifl I r Qunnamed protein product(Aspef"9ii!US orvz~ ~h1001heficalprot.in ~N5318.2WP•1"9111UO nldulans FGSC A+J

L-§~:~=~~ =~: :~=~~~~~=u~=:'Af29!1

Lr--;====================~O~lloe~'.'_l~1aily~oc~i~d SJ.onthase, puta.1il.le [foxoplasmagondiQ ~ - Oh~oPo1heticalprottinOOBOt877291Dic ..

non-ribosomal PtPfidt: ~thase MMIA ~otOCCUS KtnthUS OK 1622] MH"" rsfi9m•telluur>ntiaol

LlMA[L~MbiO.mtjuscul>l Ql nonribosomiol peptide SJ.nthetue (Bn:vibaclllus brevis]

,.-------------<>Thioesftrrtduc~ [Crocosphatl'i.mtsonli YtH 85011 poiJActtide SJ.onthase (J'IIostoc sp. PCC 7120]

CINCPB [Nostocsp. ATCC 5!1789] a COGO!I18: AcjA.COA SJ.onihefues (AMP.formint)IAMP.itdd li9i$tS 0 [...

L-----<O~:~::d:n'!fis~~;9f!tlasedomainofmutfifuncfionalnon-ribosomi.lpep

~Thloesttrreducfase(.'.nibaenlvariibilisATCC2'9+13l

poi!Jcefide S!of'lthase (Nostoc sp. PCC 7120]

-· pol!l«fide $1n1h ... ,-----------------<>unnamedprottin productrASPt1"'9illus orvziel

'

----·<,) 1--·----·-·-------·--·---·--·-·--·<> cOG102o, Non-ribosom>l peptide S~n1het... modules ond rel•t.d. '-""L ___ ~--::============:;,;:;:~,hioOOthtticll protein AN8105.2£Aspergill ...

L-0 nonribc:::~~~j~p=c~r!~~~ilfu~~

U o ~o1hetical prottin FG0239+.1 [Gibbenr:. h>oothtfic~ protein UM01697.1l\Jst

RiJPhiilt- uninold'IPltt redvcbse lCrwtococw neo1ormw "ar. neo. l:iaminoitdipate-semiaJde~e dehs.dro9enase~ putative lCrwiococc.

Oa.minoadiJ)a.tt re:cluctue enzs,.me [Ammonium.

U >? <>h-1heficolprot.in bi002611.+[Mlgnaport ... o h!oPQthetiGJ prottin FG060+1.1 [Gibberella. zeae Pt+ 1] ,-----* Ounnamed prottin productlC~dida.glibr.

n Alphumlnoodipote reductase, o.hl><es 1he reducL Q h>oothtfical protein rmrowialipoll'fiol

O~DL3+~Whbi0.90UIC>Ii~TCC 1081151 Y R~Ph•omino>dip•te reduct... tcondidulbic...

~ . ~~l>h•omlnoodipote reduct...tcindlda~bi<..

--;::====:::;:UhJo~PQtheficat proftin CIMG_01+91 [COccidioides immifis RS] L__Q. Ounnamed prottin product(l'.sp;trgiO~n o~ ~ h!tOOthetical prottin AN5610.2 (ASpergillus nh:fulans f0:5C A+J

~ alp~aminoatJiJ)U: =i~~u:nit=~iti:se=~~;;l~um chry ..

f========~~~~~~~~~~~~~Q~F"ldo~29[Ml'Coboct.riumllllumsubJp.pan.tub<rculosls K-10l H!oPOiheflc.tJ Prottin RRSL02299 [Ralstoniilsolii'IKtifllm. Bfram~o~tin MK1 S~thtfise A

pu11..tWe non-ribosomal peptide S!o-l'lfhtfase [Pseudomonas ftiJomctnSl

Figure 4.3: The Blast tree of proteins homologous to FadD9 reductase domain. The unnamed protein (highlighted in yellow) shown in the tree corresponds to the Fac:ID9 reductase domain used as a query sequence. The tree is prepared on the basis of blast pair-wise alignments and is showing nodes at maximum difference score of 0. 75.

177

Chapter4

The mycobacterial NRPS protein also contained R domains at their C-

terminus. These NRPS R domains paralogues and FadD9 paralogues from

mycobacterial genome also clustered in a close group. The NRPS R domains

showed larger sequence variation and clustered as three small groups. The

observed clustering might correspond to variation in substrate specificity or

enzymatic activity of these proteins. In our previous study (chapter 2), the

clustering observed in phylogenetic tree of mycobacterial FadD proteins

correlating with their different catalytic activities.

4.3.2 Identification of conserved motifs in FadD9 protein

All the 10 motifs characteristic of acyl-activation enzymes are conserved

in FadD9 protein. The acyl carrier protein (ACP) is a small domain and contains

phosphopantatheinylation core sequence (DxFFxLGGDSL). Phosphopantatheine

transferase enzymes catalyze the posttranslational modification of serine amino

acid residue present in this motif. The FadD9 protein paralogues showed complete

conservation ofthis motif(Figure 4.4).

gi_40796035_CAR_N_sp._NRRL_5 gi_5401549l_CAR_N_farcinica gi_l478222_FADD9_M_tb_H37Rv gi_31793774_FADD9_M_bovis_A gi_l3882413_Mtb_Mtb_CDC1551 gi_3136006_M_leprae gi_41407138_M_Av_Paratb gi_92911894_M_sp._JLS gi_l08799238_M_sp._MCS MSMEG2968 . gi_90205636_M_vanbaalenii_PY MSMEG5713

DxFPxLGGDSL RPDAHFTDLGGDSLSALSFSNL SPAAHFTDLGGDSLAALALSNL RPDAHFADLGGDSLSALSLANL RPDAHFADLGGDSLSALSLANL RPDAHFADLGGDSLSALSLANL PSNAHFTDLGGDSLSALTFGSL SPDAHFTDLGGDSLSALTFGNL RPDAHFTDLGGDSLSALTFGNL RPDAHFTDLGGDSLSALTFGNL RSDAHFTDLGGDSLSALSFSNL EPSAHFTDLGGDSLSALTFANL ASDAHFTDLGGDSLSALTLSNL

***:*******:**: : .. *

688 680 688 688 688 695 694 693 693 692 682 714

Figure 4.4: Multiple sequence alignment displays the conserved phosphopantatheineylation core sequence.

178

gi_40796035_CAR_N_sp._NRRL_S gi_S4015491_CAR_N_farcinica gi_l478222_FADD9_M_tb_H37Rv gi_31793774_FADD9_M_bovis_A gi_13882413_Mtb_Mtb_CDC1551 gi_3136006_M_leprae gi_41407138_M_Av_Paratb gi_92911894_M_sp._JLS gi_108799238_M_sp._MCS MSMEG2968 gi_90205636_M_vanbaalenii_PY MSMEG5713 gi_76809770_LtxA Gi_14210837_MxaA

R3 GDLxxPxLGLS GDIGDPNLGLD GDIGEPGLGLA GDKGEADLGLD GDKGEADLGLD GDKGEADLGLD GDKGEVDLELD GDKGEANLGLG GDKGEADLGLP GDKGEADLGLP GDKGEADLGLD GDKADADLGLD. GDIGDPNLGLT GDIGQPLLGLS GDLSQPSLGLS

* .. * *

R4 NVxGTL NWGTA NVAGTA NAAGTA NAAGTA NAAGTA NTLGTA NALGTA NALGTA NALGTA NALGTA NVLGTA NWGTA NVLGTR NVLGTS

* **

Rl VxLTGATGFLGAxLLxxLL VLLTGANGYLGRFLCLEWL VLLTGANGYLGRFLCLEWL VLLTGATGFLGRYLALEWL VLLTGATGFLGRYLALEWL VLLTGATGFLGRYLALEWL VLLTGATGFLGRYLALKWL VLLTGATGFLGRYLALEWL VLLTGATGFLGRYLALDWL VLLTGATGFLGRYLALDWL VLLTGATGFLGRYLALEWL VLLTGATGFLGRYLALEWL VLLSGANGWLGRFLTLQWL VLLTGATGFLGAYLLYELL ILLTGATGFLGAFLLEELL ***:**.*:***:* *·**

RS GYxQSKWVAExxVxxRGL GYGNSKWAGEVLLREAHD GYGNSKWAAEVLLRAAHD GYANSKWAGEVLLREAHE GYANSKWAGEVLLREAHE GYANSKWAGEVLLREAHE GYGNSKWAGEVLLREAHD GYGNSKWAGEVLLREAHD GYGNSKWAGEVLLREAHD GYGNSKWAGEVLLREAHD GYGNSKWAGEVLLREAHD GYGNSKWAGEVLLREAHD GYGNSKWAGEVLLREAHD GYAQSKWVAEKVVNIARA GYVSSKWVAEELVWVASD

**·****:.* ** *··

Chapter4

R2 VYCLVRA LICVVRG LICLVRG LICLVRA LICLVRA LICLVRA VIALVRA VIALVRA VICLVRA VICLVRA VICLVRA LICLVRA LITIVRG IYCLVRS VFCLVRS

: * : **

R6 GFLxxP QLL GLL GLL GLL GLL GLV GLL GLI GLI GLL GLL GLI GFLKA KFFPQ

Figure 4.5: Multiple sequence alignment displays the conservation of six reductase domains core motifs in FadD9 paralogues. The LtxA and MxaA sequence are included as representative NRPS R domain sequences.

The reductase domain present in NRPS proteins contain six conserved

motifs called Rl to R6 corresponding to sequences [VxLTGATGFLGAxLLxxLL],

[VYCLVRA], [GDLxxPxLGLS], [NVxGTL], [GYxQSKWVAExxVxxAxxRGL]

and [GFL:xxP] respectively (Gaitatzis et al., 2001). We performed multiple

sequence alignment of FadD9 paralogues from Mycolata genera and two

representative NRPS R domains from MxaA (siderophore myxochelin, Stigmatella

aurantiaca) and LtxA (Lyngbyatoxin, Lyngbya majuscule) proteins. The Rl to R5

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Chapter4

motifs were reasonably conserved in FadD9 protein while R6 motif was not

conserved (Figure 4.5). This analysis suggested that FadD9 R domain might have

enzymatic activity similar to NRPS R domains.

4.3.3 Characterization of FadD9 Proteins

4.3.3.1 Expression and purification of FadD9 protein

For protein expression, the pNS3 expression vector was transformed in the

BL-21 and BAP1 expression strains of E. coli to express FadD9 as apo- and bolo-

protein. The BAP1 strain expresses Sfp, a phosphopantatheinyl transferase

constitutively that could phosphopantatheinylate the serine residue of ACP

domain. The protein expression was obtained at 30°C with 0.5mM IPTG.

2 3 4 5 6 7 8 9 IO II

Figure 4.6: Purification of FadD9 protein a) SDS-PAGE gel for the Ni2+-NTA purification ofFadD9 1. Supernatant 7-9. 50mM imidazole wash 2. Wash 10. 100mM imidazole wash 3-4. 10mM imidazole wash 11. Protein marker 5-6. 20mM imidazole wash

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Chapter4

I 2 3 4 5 6 7 8 9 10 11 12 13 14 15

--

Figure 4.6: Purification of FadD9 protein b) SDS-PAGE gel for the FPLC purification ofFadD9

1. Load 8. Protein marker 2-5. Wash Fr. 7-11 9-14. 30% B-Fr. 15-20 6-7. 10%B-Fr. 13-14 15. 100% B Fr. 25

The FadD9 protein was purified from cleared cell lysate after removing

DNA by PEl precipitation. The affinity chromatography with Ni-NTA agarose

eluted most of the bolo-protein in 20mM and 50mM imidazole fractions (Figure

4.6). The apo-protein was also purified in similar manner. The protein containing

fractions were pooled and further purified by anion exchange chromatography

(ResourceQ column) using an increasing gradient of NaCl in equilibrating buffer.

The purified FadD9 proteins eluted in 300mM NaCl (30% B) containing buffer

(Figure 4.6). The chromatogram for FPLC purification, recorded at 280 nm is

shown in Appendix B. Purified proteins were flash frozen in liquid nitrogen and

stored at -70°C.

4.3.3.2 Catalytic activity of fadD9 Adenylation Domain

The enzymatic activity of FadD9 was investigated using 14C radiolabelled

lauric acid as the substrate. The adenylation domains activate carboxylic acid

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Chapter4

substrates as acyl-AMP. The acyl-adenylate is then transferred to the

phosphopantatheine arm attached to the ACP domain. We therefore performed

experiments as described in chapter 2. The FadD9 reactions were performed in the

presence and absence of coenzyme A. As shown in figure 4.7, the FadD9 protein

synthesizes acyl-CoA thioesters. This is in contrast to our earlier hypothesis that

FadD proteins present adjacent to ACP proteins would act as F AAL proteins and

activate fatty acids as acyl-adenylates.

',:~.:.···.·.·.~· ·:,·:.·.: . .-:.· ~ : • •1111-- Acyl-AMP

· 44·-.r •1111-- Acyl-CoA

· - :· ·'-~' •1111-- Origin

+ CoASH

Figure 4. 7: In-vitro assay for FadD9 adenylatio~ domain with 14C-Iauric acid. The reactions were performed in the presence and absence of CoASH. The FadD9 protein catalyzes the acyl-CoA thioester synthesis like F ACL proteins.

4.3.3.3 Catalytic activity of fadD9 ACP Domain

For the thioloation or ACP domain to be functionally active, it needs to be

post-translational modified with the phosphopantatheine (P-pant) prosthetic group.

In order to test the presence of P-pant arm, gel binding assays were performed as

182

Chapter4

described in the methods. In these assays, the holo-FadD9 protein was incubated

for 5min with radiolabelled lauric acid and ATP. If the acyl-adenylate is

transferred to the thiol of P-pant arm of ACP domain, radioactivity will be

associated with the FadD9 protein. The proteins were resolved on SDS-PAGE and

visualized using phosphorimager (Figure 4.6). Radioactive bands were observed

in reactions containing A TP suggesting that the protein synthesized acyl-AMP and

transferred it to ACP domain.

2 4

+--- Fad09 r·.:-~·x.: --~>·· .. . : . . . .

Dye Front

ATP + - + -\___) \___)

LA BA

Figure 4.6: Gel binding assay to test the function of ACP domain. Lanes 1-2 and 3-4 correspond to reaction performed with 14C-lauric acid and 14C-benzoic acid. The reactions were performed in the presence and absence of ATP. The radioactivity associated with the FadD9 protein suggests that FadD9 catalyzes the acyl-AMP biosynthesis and transfers it to P-pant arm of ACP.

4.3.3.4 Substrate specificity of FadD9 protein

Preliminary experiments with mycobacterial FadD9 revealed that lauric

acid is a good substrate. The Nocardia CAR proteins shares more than 60%

identity with mycobacterial FadD9 and is demonstrated to reduce benzoic acid

(BA) to benzaldehyde. To investigate if FadD9 can accept aromatic substrates, we

183

Chapter4

performed Radio-TLC assays with radio labelled benzoic acid. A new band at Rr

0.44 is observed in reactions performed in the absence of CoASH (lane 1, Figure

4.9). In reactions containing CoASH, a faint band of Rr 0.26 is observed. The

radiolabelled bands at Rr 0.44 and 0.26 could correspond to benzoyl-AMP and

benzoyl-CoA. These results suggests FadD9 could activate benzoic acid analogous

to nocardia CAR.

For the mass spectrometric identification of FadD9 acyl-activation domain

(A) products, the reaction components were resolved on C8 reverse phase HPLC.

A binary gradient of 10% B to 20% B in 30 min was used to elute the FadD9

products with modified BA. The elution profile was monitored at 254nm and

peaks were analyzed for the presence of acyl-AMP and acyl-CoA. As shown in the

Figure 4.8, The acyl-AMP and acyl-CoA peaks are marked in the HPLC trace.

The benzoic acid was accepted as a substrate with rather good catalytic efficiency.

2

1+--- Acyl-AMP

!+--- Acyl-CoA

1+--- Origin

+ CoASH

Figure 4.9: Radio-TLC assay of FadD9 with benzoic acid. Lanes 1 and 2 correspond to the FadD9 reaction performed with and without CoASH. Additional radioactive bands are observed in these reactions and could be benzoyl-AMP and benzoyl-CoA.

184

Chapter4

In chapter 3, we have explored the promiscuous nature of substrate

specificity of mycobacterial F ACL proteins. Our results showed that several

modifications distal to the a-carbon are tolerated with reasonable efficiency. In

order to study the aromatic binding site, we used a library of hydroxyl, amino and

phenyl-modified aromatic carboxylic acid substrates. Formation of acyl-AMP and

acyl-CoA thioesters was examined on analytical HPLC and elution profile was

monitored at 254nm (Figure 4.1 0). The peaks other than the void volume were

collected and analyzed by mass spectrometry for the presence of acyl-adenylates

and acyl-CoA molecules.

The FadD9 showed a good catalytic efficiency with >70% of benzoic acid

converted to acyl-AMP and acyl-CoA. The relative proportion of acyl-AMP and

acyl-CoA was similar to the results obtained with F ACL proteins, where acyl-CoA

is seen as the primary reaction product with acyl-AMP is <1% of total reaction

products. The FadD9 synthesized equal amounts of acyl-AMP and acyl-CoA

thioester with 4-hydroxyl-benzoic acid. This acid was not good substrate and only

5% of the acid was converted into product. The 2,4-Dihydroxy-, 2,6-Dihydroxy­

and 3,5-Dihydroxy benzoic acid were tested as FadD9 substrates. No activity was

observed with 2,4- and 3,5- modified acids and only weak activity was observed

with 2,6-dihyroxy benzoic acid.

The benzoic acids modified with amino modification were better substrates.

The 4-amino and 3,5-Diamino modified benzoic acid were better substrates.

Interestingly, >80% of these modified acids were converted to reaction products

with acyl-AMP being the major product. Less than 1% acyl-CoA was observed in

4-amino-benzoic acid while no acyl-CoA could be detected with diamino modified

185

Chapter4

benzoic acid. We tested 4-hydroxy-biphenyl acid (BPA) as substrate for FadD9.

The BPA acid and acyl-AMP coeluted at 27min and acyl-CoA thioester eluted at

29min. The enzyme activity with BPA acid followed the trend observed for amino

modified benzoic acid and acyl-AMP was the major product (90% of total). The 4-

hydroxyl-biphenyl-CoA could also be detected. These peaks were collected and

analyzed by nano-spray ESI-MS.

The various different peaks were collected from the HPLC elution of

reaction products and were analyzed for presence of acyl-AMP and acyl-CoA. The

figure 4.10 to 4.13 display the tandem MS spectra obtained for FadD9 products

with benzoic acid, 4-hydroxy-benzoic acid, 3,5-diamino benzoic acid and 4-

hydroxy-biphenyl acid. As described in the chapter 2, the acyl-AMP fragments at

the labile phosphoester linkage and generates the AMP moiety seen as 346.04 peak

in the spectra. Dehydration of this peak is also a common fragment observed at

328.07. Increase in collision energy results in further fragments the AMP moiety

and 134.06 peaks is generated corresponding to the adenine moiety.

Table 4.2 Lists the different elution conditions used to resolve the acyl-AMP and acyl-CoA thioesters on C5 reverse phase-HPLC column.

186

Chapter4

Retention time (min) Carboxylic acids Elution Gradient

Acyl-AMP Acyl-CoA

Lauric acid !socratic: 70%B 8.0 8.5

Benzoic acid (BA) 15.8 17.5

4-Hyd.roxy-BA 13.1 ND

2,4-Dihydroxy1-BA ND ND

2,6-Dihyd.roxyl-BA Binary: 8.7 ND O.Olmin: l0%B, 30min:

3,5-Dihyd.roxyl-BA 20%B, 40min: 100%B, 8.4 ND

4-Amino-BA 5.5 6.5

3,5-Diamno-BA 11.7 12.5

4-Hyd.roxy-biphenyl acid 29 30

The acyl-CoA thioester product for these four substrates were also analyzed

by ESI-MS. The benzoyl-CoA molecular mass was calculated to be 886.06 and

tandem MS of this peak generated many fragments characteristic of acyl-CoA

thioesters. As discussed in chapter 2, dephosphorylation of the 3 'phophodiester

bond is the most common fragment ion and generates a [M-80r1 ion (fragment ion

2). The other two phosphor-diester linkages are also labile and fragment to

generate fragment ions 3-5. The fragment ions 2 and 5 retain the acyl-group.

Comparison of acyl-CoA spectra further validated this fragment assignment as the

fragment ions 2 and 5 show change in mass in accordance with the carboxylic acid

starter used to synthesize acyl-CoA.

187

~ ~ .,J .5 a; a:

-~ ~ .,J

.5

~

100 134.06

90

80

70

60

50

~

30

20

MS/MS Fragmentation Pattern of 450.11

2 328.07

[M-H]-1

10 I I 4i.11

O ~10~0~~1~~--~-18~0~--2~20--~-26~0~--3~00--~-M~0~--3~80--~-42~0~~~~0--~-50~0--~

mlz, amu

MS/MS Fragmentation Pattern of 434.57 NH,

4 "JC" o• 0< r ' " . C(ls~~Y"-'~Y.x' \? _\? ~1 ~,·· o-~ lj'- 0 0 0 0- '

H o\1 4 c;;;;::o.-2

100 408.01 5 6

90

80

70

60 5

50 523.06

~ 3-H20

1 443.10 272.95 328.04

28;0 [M-H]-1 30 134.22

Los 20 158192

2 T 3 s-P04 790118 10 30

r~l .I 6\11 686(1 0

j 385i~ h 150 250 350 450 550 650 750 850

mlz, amu

Chapter4

Figure 4.10: Tandem mass spectrometric analysis of benzoyl-AMP and benzoyi-CoA tbioester molecules. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

188

100

90

80

70

~ 60

,.J 50 .E

~ 40

30

20 1i04 10

100 150

100

90

80

70

~ 60 134.03

,.J 50 .E

~ 40

30

20

10

0100

MS/MS Fragmentation Pattern of 466.06

N-.1: 0 ~ JL) ~ 0 N N

)J tt·o---, -~1 HO 2 a

2 346.04

2-H20

T 4i00

200 250 300 350 400 450

mlz, amu

MS/MS Fragmentation Pattern of 450.59 NH,

06 OH r4 3 ti:J

~~~'1("'-'~Jx'o-'i/ 'i/~N1 )J I" 0 o ,,. 6 6: 0

~ H ~

~2 339.04 5 6

357.05

4 408.01

mlz, amu

[M-H]-1

500 550

[M-H]-1 886.06

600

900 1000

Chapter4

Figure 4.11: Tandem mass spectrometric analysis of 4-hydroxyl-benzoyi-AMP and 4-hydroxyl-benzoyi-CoA thioester molecules. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

189

Chapter4

MS/MS Fragmentation Pattern of 480.14

N~~ '91 0 {N 1

j H,N 0-~-~1

0-

NH, "oH ol'l

100 328.07

2·H20 90

80

70 - [M·H]·' ~ 60

.I 50 480.14

.5

~ 40

30

20 193.01

10 1T6 l 2 0 I

50 100 150 200 250 300 350 400 450 500 550

rnlz, amu

MS/MS Fragmentation Pattern of 449.61 NH,

6 4 3 tJCN

~""o"f~ H,N ~NY"'--'N~ " " 1 __ ... o-, \"0 0 0 0 0-

H Q\1 NH, O"P-0- 2

100 449.61 5 0

90

80

70

~ 60

.I 50 4 2

.5 408.04 820.24

~ 40

="'ft:·,t 30

20

·34r6 3·H20

7r7 491.17 10 r 553.14 r 765.13 0

100 200 300 400 500 600 700 800 900 1000

rnlz, amu

Figure 4.12: Tandem mass spectrometric analysis of 3,5-Diamino-benzoyi-AMP and benzoyi­CoA thioester molecules. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

190

-~ ... .: ~

-:::li! e.. ... .: ~

100

90

80

70

60

50

40

30

20

10

0 50

MS/MS Fragmentation Pattern of 542.12

~~~ 02-o-r~1

"oH o\:l HO b [M·H]-1

542.12

134.,5016

100 200 300 400 500

mlz, amu

MS/MS Fragmentation Pattern of 480.59 NH, 4 Ni:N " 007. "· ~~~Y"-'~Yx' ~-i)-ijw~

o o' o-o-

" ~ o=P-o-1 H 5 o

100 134.05

90 4

80 408.02

70 3-H20 535.13 5

60 615.11

50 346.06 3

40 307.05 553.15

30 272.97

58.93

,1IJI. 426.03

20

L rLj II 10 l.t. j I I I

0 100 200 300 400 500 600 700

m/z, amu

Chapter4

600 700

2 882.19

I 800 900 1000

Figure 4.13: Tandem mass spectrometric analysis of 4-hydroxyl-bipenyi-AMP and 4-hydroxyl-bipenyi-CoA thioester molecules. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

Combination of HPLC and ESI-MS analysis confirmed that FadD9 accepts

medium chain fatty acids as well as aromatic acid. This substrate promiscuity is

even larger than the FACL proteins FadD19 and FadD6 (described in chapter 3).

191

Chapter4

4.3.3.5 Catalytic activity of fadD9 Reductase Domain

To investigate the reductase domain activity, the FadD9 assays were

performed with 14C-radiolabelled lauric acid and benzoic acid in the presence and

absence of NADPH. The reactions were incubated for 30min and quenched by

adding 1 O)ll of 10% glacial acetic acid. The reaction products were extracted with

ethylacetate (EtoAc) and dried under vacuum. The product was resuspended in

1 O)ll of EtoAc and spotted on TLC. The TLC was developed in 90% hexanes in

EtoAc along with authentic chemical standards for lauroyl-alcohol and lauroyl-

aldehyde (Rr 0.45 and 0.85 respectively).

LAid

BAld LA •

- BA

• ~ • -- • NADPH + +

Figure 4.14: Radio-TLC investigations of FadD9 reductase domain activity. The reactions were performed in the presence and absence ofNADPH. New bands of ~0.9 and 0.8 Rr were observed in lauric acid and benzoic acid reactions performed with NADPH.

The results of FadD9 reactions are shown in figure 4.14. The FadD9

protein synthesized a new product on addition of NADPH. The Rr of radioactive

band was found to be 0.8. Since nocardia CAR produces aldehyde, we

192

Chapter4

hypothesized that FadD9 will also synthesize aldehyde. The observed difference in

Rr needs to be validated by HPLC and mass spectrometric analysis.

4.3.3.6 LC-MS investigations of FadD9 R domain reaction

products

To identify the FadD9 catalytic products, mass spectrometric analysis were

performed. The lauroyl-aldehyde standard was procured from sigma and analyzed

by electrospray ionization mass spectrometry (ESI-MS). This compound did not

ionize well and reasonable spectra could be obtained with sample concentration in

millimolar range. The compound formula of lauroyl-aldehyde is C12H240. The

expected molecular mass ofthis compound is 184.3. In the positive ion mode, this

compound is expected to yield molecular .masses of 185.3 corresponding to

[M+Ht1 ions. The Tof-MS spectra revealed a molecular ion peak of 184.3. The

tandem MS of this peak did not yield any specific fragmentation pattern. In

addition, increasing the collision energy reduced the height of this peak but few

daughter ions could be observed. The peak height showed a proportional increase

with increase in lauroyl-aldehyde concentration.

The most common fragmentation pattern for fatty aldehydes is a-cleavage.

This is a rearrangement of the fatty aldehydes longer than six carbons, where in the

loss of aldehyde hydrogen generates a positively charge ion. This ion is stable and

does not fragment further. We therefore, believe that the electrospray ionization

leads to the alpha cleavage of lauroyl-aldehyde. As the lauroyl-aldehyde does not

ionize efficiently in ESI-MS, it will be difficult to detect products synthesized by

FadD9 enzyme. We therefore decided to use 14C-radiolabelled substrates, the

193

Chapter4

concomitant change in concentration of the FadD9 product would facilitate

characterization.

Mass Spectrometric Analysis of Lauroyi-Aidehyde

~· + . _ _ ~ I a-Cleavage '"'-./"H6V/ H

0+

~I 6

184.22

185.21

183.18

184.0 186.0 188.0 190.0 192.0

m/z, amu

Figure 4.15: ESI-MS investigations of FadD9 reductase domain activity. The Tof-MS spectra's of lauroyl-aldehyde standard and 8.3min peak of FadD9 reaction are shown in panel A and B. The isotopic profile of standard lauroyl-aldehyde revealed the expected isotopic distribution.

+-LA ~0.3

~ 0.2 J9 < 0 E >

50 0.1

0 0

0 2 4 6 8 10 12 14 16 18 20

Minutes

Figure 4.16: HPLC investigations of FadD9 reductase domain activity. The chromatographic traces of FadD9 reaction products and lauric acid-lauroyl-aldehyde mixture are overlayed. The FadD9 reactions were performed with 14C-radiolabelled lauric acid with 2mM NADPH. In this system, lauric acid and lauroyl-aldehyde elutes at 5.2 and 8.3min respectively. A radioactive peak eluted with retention time same as lauroyl-aldehyde as shown in the box.

194

Chapter4

Mass Spectrometric Analysis of FadD9 Product

183.06

1.00

0.80

~ 0.60 ..., .5

~ 187.08

0.40

182.03 184.07 185.07

0.20

A .h * 188.08

181.03 186.07

~ 0.00 k ~ 181.0 182.0 183.0 184.0 185.0 186.0 187.0 188.0

rnlz, amu

Figure 4.17: The TOF-MS analysis of radioactive product: The Tof-MS spectra of 8.3 min peak shows a marked increase in the 186.07 e4C isotope) as compared to 184.3 e2c isotope). The 183.06 is a background peak and 13C isotope peak might be contributed by 14C-isotope of this contaminant.

The enzymatic reactions of FadD9 were performed with 14C-radiolabelled

lauric acid for 3hr. The reactions were quenched and extracted with ethylacetate.

The residue after vacuum concentration was resuspended in methanol and loaded

on C8 reverse phase column. The reaction products were resolved by isocratic

gradient of 10% dichloromethanol in methanol and eluents were passed through an

in-line radioactive det~ctor. This solvent system resolve lauric acid and lauroyl-

aldehyde and both these compounds elute at 5.2min and 8.3min respectively. In the

FadD9 reaction run, a small radioactive peak eluted with retention time same as

lauroyl-aldehyde (Figure 4.16, shown in the small box). This peak was dried in

vacuum concentrator and the residue was resuspended in 90% ethanol containing

traces of ammonium acetate solution. The Tof-MS profiles of 1mM lauroyl-

aldehyde standard and 8.3min HPLC peak are shown in figure 4.17. It can be

observed that peak corresponding to the 14C-isotope (186.03) was considerably

more populated than could be attributed to the natural distribution carbon isotopes.

195

Chapter4

This shift in the isotopic profile confirmed the identity of FadD9 reduced product

as lauroyl-aldehyde.

FadD9

Fatty Acid Fatty Aldehyde

Z-Lys-Me l

Acylated Z-Lys Me Schiff base adduct

Figure 4.18: Schematic representations of the strategy used for peptide adduct formation. The aldehyde reacts with free amine and leads to formation of Schiff base. NaCNBH3 reduces this labile adduct to stable saturated CH2-NH2 linkages.

Since the aldehyde did not ionize well in ESI-MS, we further improved on

our detection technique by modifying the aldehyde products. The free aldehydes

synthesized by FadD9 were reacted with the benzyloxycarbonyl lysine methyl

ester (z-Lys-OMe). The a-amino and carboxylic acid functional groups of lysine

are modified by acylation with benzyloxycarbonyl and methyl ester respectively.

Thus, this substrate mimic posses only the s-amino group as free functional group

that can react with aldehyde moieties. The aldehydes are reactive molecules and

react with free amino groups to synthesis a Schiff base adduct. The schiff base

adduct is unstable at pH 7.0. Therefore mild reduction with gentle reducing agents

196

Chapter4

like NaCNBH3 was performed to convert the Schiff base adduct to stable amino

alkylated compounds (Fenaille et al., 2003). The schematic representation of

conversion of aldehyde to stable amino-alkylated z-Lys-OMe is displayed in figure

4.18.

The FadD9 reactions were processed as described earlier for HPLC

analysis. After the vacuum concentration, the residue was resuspended in 70%

ethanol and incubated with z-Lys-OMe and NaCNBH3 for 30min. The reaction

was extracted with 400Jll ethylacetate and dried again. The residue was

resuspended in 90% ethanol and analyzed by nano-spray ESI-MS. For the reaction

performed with lauric acid, the TOF-MS spectra revealed the major peak at 463.36.

This peak corresponds to the [M-H] l- ion of reduced lauroylaldehyde adduct of z­

Lys-OMe. Tandem MS analysis of 463.36 peak revealed several fragments that

could be mapped to dodecanyl-z-Lys-OMe, The most prominent peak observed

was the [M-44] peaks that could arise by neutral loss of C02• The most labile

linkages in this molecule are the ester and peptide linkages and fragment ions 1-3

result from the breakage of these bonds. Increase in collision energy generated

additional fragments and fragment 4 could be attributed to amino-alky-side chain

of lysine moiety. Adducts ofFadD9 products synthesized with various carboxylic

acids like palmitic acid, benzoic acid (BA), 4-hydroxyl-BA, 3,5-diamino-BA, 2,4-

dihydroxy-BA, 4-amnio-BA, 4-hydroxy-biphenyl acids etc were also analyzed by

mass spectrometry. The tandem mass spectrums of these adducts are shown in

figure 4.19- 4.22.

197

A

32

28

24

~ .,I 20 .E 'ii 16 a::

12

8

4

0100

B

100%

90%

80%

~ 70%

.,I 60%

.E

~ 50%

40%

30%

20%

10%

0%100

142.09

144.10

MS/MS Fragmentation Pattern of 463.36

\

0~/--.. /"'-._ -~ 4'f--"'~

____ N~-

_Q.~-- ·- 3 __ 0

o_ ... --2

·- 1

I"" -6

Chapter4

[M+H]•• 463.36

3 2 [M-C02H]•1 174.13 4 276.29

·5.rlT3 3T:T·

I .ol.

140

252.28 l 23tiJ

3T·1 371r30

180 220 260 300 340 380

mlz, amu

MS/MS Fragmentation Pattern of 519.43

\

0~~ /"'-._ /"'-._ -~ 4-f--~~

NH -o"5:---. 3

===o-~=-- 2

I"" -6

·- 1

265.20

234.16 I 25ly•sl

309.19

T 2

3 411.37 1 383.37 " •

332.34 ~ ~I 4271.36 'T. I -1. I IL

140 180 220 260 300 340 380 420

mlz, amu

41i36

420 460 500

[M+H]•• 519.43

460 500 540

Figure 4.19: Tandem mass spectrometric analysis of FadD9 synthesis aldehydes with lauric acid and Palmitic acids is shown in panel A and B. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

198

A

100

90

80

;e 70

~ 60 ..J .E 50

;i 40 142.09

30

20

10 114.10

0 120

B

100

90 107.06

80

70

;e 0 60 -..J 50 .E 144.11

;i 40

30

20 14210

10

qoo 140

MS/MS Fragmentation Pattern of 385.23

181.11

3 23416 24~.17

. ,I I 160 200 240 280 320 360

mlz, amu

MS/MS Fragmentation Pattern of 401.25

\

O~~o-OH NH -ci§;: ____ 3

---- o"'•. 2

---~--- 1

u 295.18

[M+H]•1

251.19 401.25

4 2i16

2 [M-C02H]•1 293 8

19,.13 35j.24

180 220 260 300 340 380 420

mlz, amu

Chapter4

[M+H]•1

385.23

400

460 500

Figure 4.20: Tandem mass spectrometric analysis of FadD9 synthesis aldehydes with benzoic and 4-hydroxyl-benzoic acids is shown in panel A and B. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

199

A

B

~ ~ .. .E

~

MS/MS Fragmentation Pattern of 415.28

~NH-cf~ NH N~ --Q§!--- 3 ----- o'·· 2 ---0-- 1

[M+H)•1 415.28

100

90

80

70 121.09

~ ~ 60 .. .E 50

'ii 40 a: 30

20

211.15 [M-C02 H)•1 3 2 153.05 279.19 371.27

307.21 10 194.12 0100

13~ 14 l I I 38j25 I. I

140 180 220 260 300 340 380 420

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

O% 100

mlz, amu

MS/MS Fragmentation Pattern of 417

144.11

123.05 II J .II /58.,13 r.'"''2 120 140 160 180 200

251.18 234.16258.17

.. JJ "'

[M+H]•1

417.25

[M-C02H]•1

\ [M-H20)+

301::,1 [327122 37r613i.2.

3 295.18

2

220 240 26&,,.~gg,u 300 320 340 360 380 400 420 440

Chapter4

Figure 4.21: Tandem mass spectrometric analysis of FadD9 synthesis aldehydes with 3,5-diamino- and 2,4-dihydroxyl-benzoic is shown in panel A and B. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

200

A

16.0

14.0

12.0

~ 10.0 .oJ .E 8.0

&! 6.0

4.0

2.0

0.0

B

100

90

80

70

;;e 60 e... .oJ 50 .E

&! 40

30

20

10

0100

123.10

I 144.11

II 120

142.09

1 .10

MS/MS Fragmentation Pattern of 400.25

[M+H]•1

400.25

2 292.19

182.12

3 [M-C02H]•1 295.18 166.09 264119

II ~ 160 200 240 280 320

rnlz, amu

MS/MS Fragmentation Pattern of 521.

\ 0

~~NH-o--o-OH ---_:;~t!. • .Q.'=',--•• 3

···c/:

356.25

I 360 400

[M+H]•1

521.40

[M-C02H)•1

477.41

234.15 334.31 3

1 41~.34 1

17i.13 h26 31~31 t 38534~~ 36r J2t

140 180 220 260 300 340 380 420 460 480 520

rnlz, amu

Chapter4

Figure 4.22: Tandem mass spectrometric analysis of FadD9 synthesis aldehydes with 4-amino-BA and 4-hydroxy-biphenyl acids is shown in panel A and B. The chemical structure of the molecule with the expected fragmentation profile has been drawn beside the spectra.

201

Chapter4

4.3.3.8 Kinetic analysis of FadD9 activity

To study the reaction mechanism, the time kinetics were performed for

FadD9 aldehyde product for lauric and Palmitic acid. A linear increase in the

product formation was observed till -8min (Figure 4.23), reached saturation after

that. As the steady state kinetic parameter need to be calculated in the linear range

of product formation time scales, we therefore chose 5min as the incubation time

for KM calculation assays.

A

2 3 4 5 6

---+ Time

c

12

u; Cll 0 8 e .:.. , 4 < ...I

0 0 20 40 60

Time (min)

B

__,.LAid

__,.LA

D

u; .!! 0 e .:.. , ~

3

2

0 0

2 3 4 5 6

__,. PAid

__,. PA

20 40 60

Time (min)

Figure 4.23: Time kinetics of FadD9: Panel A & B correspond to Radio-TLC assays performed for LA and PA. The Lanes 1-6 of panel A and B correspond to 2, 5, 10, 30 and 60min incubation times. C and D display the product formation profile in relation to incubation time for both these substrates respectively. All the kinetic data presented here are means (n = 3) of three independent experiments with SE values <15%.

202

A

B

c

2 3 4 5 6 7

....... +-L-Aid

·······~LA

2 3 4 5 6 7

.... " ~- -~ ~-· .,_ P-Aid

••••••• ._PA ·'. ~· · .. ·'. ..,.~:: . :-·•, . ·~: :. ;f~

. . :~

.. -:•

2 3 4 5 6

·~~if: .... ~ '·~~". '.-.i~k . . ··.~~:::'::.~,. .. :·:: ~.:·;.,:' .. ·~; .. : :.·~·:_:;·_

2400

r:: ~·e 1600 ::::1-'01/1 o.!! ... 0

800 o..e S:

0 0

300 c

~ ·e 200 ::I iii

'CCI) o-... 0 o..e 100

S: 0

350

c 250 ~ ·e ::::1-'0"' o.!! 150 ... 0 o..e

S: 50

Chapter4

KM= 98.3 ± 39.371JM

250 500 750 1000

LA (JJ.M)

KM = 103.8 ± 22. 71JM

0 250 500 750 1000

PA (JJ.M)

0 150 300 450 600 750

BA (JJ.M)

Figure 4.24: Steady state kinetics of FadD9 (KM for Lauric acid, Palmitic acid and benzoic acid): The Lanes 1-7 ofTLC in A and B contain 5, 15, 35, 75, 150, 300 and 700 1-1M of LA and PA respectively and the lanes 1-6 in Panel C contain 15, 35, 75, 150, 300 and 700J.1M of benzoic acid. The steady state kinetics curve obtained for these three substrates are shown next to the Radio-TLC images. All the kinetic data presented here are means (n = 3) of three independent experiments with SE values <15%.

The apparent KM values for lauric, Palmitic and benzoic acids were

estimated by carrying out competitive experiments with lauric acid (Figure 4.24).

In these experiments, the 14C-radiolabelled substrate used was lauric acid and

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different concentration of lauric, Palmitic and benzoic acids were added to separate

reaction. The concentrations were varied between 5, 15, 35, 75, 150, 300 and

700J.LM. The LA, PA and BA KMwas estimated to be 91.67, 118.5 and 357.9J.LM

respectively. The catalytic turnover numbers for lauric, palmitic and benzoic acids

were estimated to be 3911.2, 4794.3 and 1720.4 sec-1 M-1 respectively. These

results suggest that FadD9 displays a robust activity towards these carboxylic acid

substrates. Thus we have characterized FadD9 as a carboxylic acid reductase that

reduces both alkyl and aromatic carboxylic acids to their corresponding aldehydes.

4.4 DISCUSSION

The FadD9 showed homology with nocardia carboxylic acid reductase, a­

aminoadipic acid reductase (AAR) and reductase domains present at the C­

terminus of PKS and NRPS proteins. Both AAR and PKSINRPS reductase

proteins reduce carboxylic acids to their corresponding aldehydes by using one

molecule of A TP and NADPH. These aldehyde products could either be

transaminated directly or further reduced to alcohols. Interestingly, the NRPS

reductase domain from myxochelin biosynthesis pathways can perform two rounds

of iterative reduction and synthesize alcohol. Combination of LC-MS studies

unambiguously identified FadD9 protein as carboxylic acid reductase (CAR) that

reduces carboxylic acids to their corresponding aldehydes products. Substrate

specificity studies revealed that FadD9 accepts a large repertoire of carboxylic

acids including methyl, amino or hydroxyl modified straight chain alkyl and

aromatic acids. We systematically characterized the A and R domains products

with these modified carboxylic acids. In addition, we improved the sensitivity of

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our detection system by analyzing aldehyde products as adduct of peptide mimic,

benzyloxycarbonyllysine methyl ester (z-Lys-OMe).

0

R~OH FA

\, sr t«rrr 1

R <~

+-­NADPH

1 0

R~s

(_ A'f}.---R ~(~ Figure 4.25: Schematic representation of FadD9 reaction mechanism.

On the basis of our systematic characterization of individual domains, we

could now propose a mechanism of carboxylic acid reduction as shown in figure

4.23. The acyl-activation (A) domain activates carboxylic acids as acyl-adenylates.

These acyl-adenylate intermediates are thioesterified to the sulfhydral group of

phosphopantatheine (P-pant) arm attached to acyl carrier protein (ACP, T). This

thioester bond is then reduced to aldehydes by reductase (R) domain. Since the R

domain reduces P-pant thioester of carboxylic acid substrate to aldehyde products,

we investigated if acyl-CoA thioesters could be used as substrates directly. No

activity was obtained with acyl-CoAs suggesting that P-pant attached substrates

are required for catalysis.

205