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Biological Chemistry

Biochemical characterization of an S-adenosyl-L-methionine


dependent methyltransferase (Rv0469) of Mycobacterium
tuberculosis

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Journal:

Manuscript ID:
Manuscript Type:

Complete List of Authors:

23-Jan-2013

Meena, Laxman; CSIR-Institute of Genomics and Integrative Biology,


Allergy and Infectious diseases
Chopra, Puneet
Vishwakarma, Ram
Singh, Yogendra
Membranes, Lipids, Glycobiology

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Keywords:

Research Article

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Section/Category:

BIOLCHEM-2013-0126

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Date Submitted by the Author:

Biological Chemistry

Tuberculostearic-acid, S-adenosyl-L-methionine, Mycobacterium


tuberculosis, Oleic-acid, methyltransferase, Fatty-acid

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Biological Chemistry

Biochemical characterization of an S-adenosyl-L-methionine dependent

methyltransferase (Rv0469) of Mycobacterium tuberculosis

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Laxman S. Meena*, Puneet Chopra, Ram A. Vishwakarma and Yogendra Singh

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CSIR-Institute of Genomics and Integrative Biology, Council of Scientific and

Industrial Research, Mall Road, Delhi-110007, and

Bio-organic Chemistry Lab, National Institute of Immunology, New Delhi

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Running Title: Biosynthesis of Tuberculostearic acid

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*To whom reprint request should be addressed:

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* Dr. Laxman S. Meena, Ph.D


CSIR-Institute of Genomics and Integrative Biology
Mall Road, Delhi-110007
Telephone no: 011-27666156
Fax No: 011-27667471
E-mail ID: meena@igib.res.in
Laxmansm72@yahoo.com

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Biological Chemistry

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Abstract

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Tuberculostearic acid (l0-methylstearic acid, TSA) is a major constituent of

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mycobacterial membrane phospholipids and its biosynthesis involves direct methylation

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of oleic acid esterified as a component of phospholipids. Methyltransferases of

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mycobacteria were long proposed to be involved in the synthesis of methyl-branched

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short chain fatty acids, but direct experimental evidence is still lacking. In this study, we

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identified methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a

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novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase capable of

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catalyzing the conversion of olefinic double bond of phospholipid linked oleic acid to

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biologically essential tuberculostearic acid. Therefore UmaA catalyzing such

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modifications offer viable target for chemotherapeutic intervention.

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Key Words: Fatty-acid, S-adenosyl-L-methionine, Mycobacterium tuberculosis,

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methyltransferase, Tuberculostearic-acid, Oleic-acid,

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Abbreviations Used: PC, 1, 2 Dioleoyl-sn-Glycerol-3-phosphocholine; PE, 1, 2

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Dioleoyl-sn-Glycerol-3-phosphoethanolamine; PS, 1, 2 Dioleoyl-sn-Glycerol-3-

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phosphoserine; SAM, S-adenosyl-L-methionine; TSA, Tuberculostearic acid

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Biological Chemistry

Introduction

An important key to the success of pathogenic mycobacteria is its unusual cell

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wall architecture. The characteristic cell wall core is composed of arabinogalactan-

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mycolate layer covalently linked to the cell wall peptidoglycan (Dmitriev et al., 2000),

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phosphatidylinositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan

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(LAM). The glycolipids derived metabolically from phosphatidylinositol (PI) are the

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prominent interspersed phospholipids/lipoglycans of mycobacterial cell wall (Besra et al.,

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1997). They remain non-covalently attached to the plasma membrane through their

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phosphatidyl myo-inositol anchor. Together, this highly complex array of lipids and

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glycolipids form a thick barrier and protect mycobacterium from noxious chemicals as

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well as during host infection. Therefore, the enzymes involved in the biosynthesis of this

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essential structural component of M. tuberculosis H37Rv (Mycobacterium tuberculosis

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H37Rv) offer a potential target for the chemotherapeutic intervention.

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Among the potentially attractive drug targets are the enzymes involved in the

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synthesis of the main mycobacterial phospholipids (Goren et al., 1984).

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Phosphotidylinositol (PI) is an essential phospholipid of mycobacteria (Jackson et al.,

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2000) as it constitutes a lipid anchor to the cell envelop for PIMs, LM and LAM. The sn-

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1 and sn-2 positions of PI are acylated by C-16 and C-19 fatty acids respectively (Nigou

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et al., 1997). The fatty acid at sn-2 position represents C-19 monomethyl-branched

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stearic acid, Tuberculostearic acid (TSA). Tuberculostearic acid arises by methylation of

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oleic acid esterified to phospholipids (oleyl-PL), with S-adenosylmethionine (SAM) as

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the methyl donor. Oleic acid is first alkylenated at C-10 position to give 10-methylene

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stearic acid, which is subsequently reduced to 10-methylstearic acids with NADPH as a

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cofactor (Akamatsu et al., 1970). Phetsuksiri et al., in their work on thiourea isoxyl

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(ISO), a frontline anti-tuberculosis drug, identified the synthesis of oleic acid as the

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primary target of ISO (Phetsuksiri et al., 2003). The authors also observed a dramatic

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effect of ISO on the synthesis of tuberculostearic acid as a consequence of its effect on

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oleic acid synthesis. The formation of TSA bears a strong resemblance to the enzymatic

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modification of mycolic acids in M. tuberculosis. The double bonds in their

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meromycolate chain are modified with cycopropane rings and methyl branches through

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the action of a large family of SAM dependent methyltransferases (Takayama et al.,

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2005). Previous studies have established these highly homologous methyltransferases to

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be functionally distinct. In a study, Grzegorzewicz et al., demonstrated that treatment of

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M. tuberculosis with Isoxyl (ISO) and thiacetazone (TAC) inhibit the dehydratase step of

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the fatty-acid synthase type II elongation cycle (Grzegorzewicz et al., 2012). Two

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additional methyltransferases were also identified as the members of SAM dependent

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methyl transferases family and were annotated as umaA and umaA2 (Cole et al., 1998).

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Whereas umaA2 (PcaA1) was later characterized as a cyclopropane synthase, however,

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umaA2 has not been biochemically characterized and its function is still not clear.

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However, in a studs, Laval et al., showed that disruption of umaA in M. tuberculosis does

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not have any effect on composition of short chain fatty acids or mycolic acids (Laval et

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al., 2008).

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In the present study we cloned, expressed and purified UmaA and investigated its
function and established it as a SAM dependent methyltransferase responsible for the

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modifications of short chain fatty acids. We demonstrate that UmaA is capable of

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catalyzing the conversion of oleyl-PL to tuberculostearic acid in vitro. Thus UmaA

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represents a family of methyltransferases involved in the biosynthesis of branched-short

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chain fatty acids.

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Biological Chemistry

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Results and Discussion

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Methyltransferases of M. tuberculosis represent a large family of highly

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homologous proteins involved in enzymatic modification of mycolic acids. The double

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bonds in meromycolate chain of mycolic acids are catalyzed to cycolpropane ring and

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methyl branches by the addition of methyl group derived from SAM. umaA shares

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striking amino sequence similarity with the members of this gene family. Therefore, it

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was pertinent to see whether umaA encodes a functional methyltransferase. In this study,

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umaA gene was cloned in an E. coli expression vector, pGEX-5X-3. Over expression of

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this protein resulted in a fusion protein of appropriate molecular weight of 59 kDa (33

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kDa UmaA + 26 kDa GST tag) on a 10% SDS gel. Produced protein (UmaA) was

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purified to homogeneity as GST fusion protein (Fig 1A).

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The hydropathy profile predicted UmaA as a soluble protein. To confirm

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bioinformatics prediction, sub cellular fractions of M. tuberculosis was prepared by

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ultracentrifugation and purity of each sample was determined by checking specific

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markers of that particular cellular fraction. Sub cellular fractions were separated by SDS-

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PAGE and western blotting was done using UmaA antisera. UmaA was detected as a

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33-kDa protein in the whole cell lysate and cytoplasmic fractions and was absent from

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both cell wall and cell membrane fractions (Fig 1B). These results confirmed that UmaA

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is a cytoplasmic protein of M. tuberculosis H37Rv.

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To biochemically characterize UmaA as a methyltransferase, a standard protocol

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was followed (Yuan et al., 1998) to determine the optimal in vitro conditions. An initial

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assay was performed with M. smegmatis crude lysate in the presence of radiolabelled

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[methyl-3H] SAM as methyl group donor. After saponification and methyl ester

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preparation, the extracted products were analyzed on a silica TLC plate. In this study, the

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majority of the label was transferred to fatty acid methyl ester (FAME) fractions,

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representing short chain fatty acids. However, traces of radiolabel was also observed in

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the mycolic acid methyl esters (MAMEs) fraction, representing the long chain fatty

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acids (Fig 2A, Lane 1). To determine the specific activity of purified UmaA, the activity

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of endogenous enzymes of M. smegmatis was eliminated by heat inactivation of crude

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lysate at 90 for 10 min (Fig 2B, Lane 1). Under similar reaction conditions both heat

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treated (HT) and non heat treated (NHT) M. smegmatis crude lysates were incubated with

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crude extracts of E. coli cells overexpressing UmaA or purified UmaA in the presence of

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radiolabelled SAM. In parallel control reactions with crude extract of the strain of E. coli

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containing empty vector pGEX-5x-3 was also performed. Interestingly, the specific

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labeling of FAMEs in HT (Fig 2B, Lane 2) and a substantial increase of radiolabel in

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FAMEs fraction of NHT samples (Fig 2A, Lane 2) were observed with E. coli cells over-

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expressing UmaA. In the same study, we also observed that both anti-UmaA antibody

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and S-adenosyl-L-homocysteine, a non-methylated analog of SAM completely abrogated

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the methyl transfer. (Fig 2A, 2B, Lanes 3 and 4, respectively). Under similar condition,

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purified UmaA protein also resulted in the specific labeling of FAMEs in HT Fig 2C,

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Lane 1 and a substantial increase of radiolabel in FAMEs fraction of NHT samples (Fig

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2D, Lane 1). In the same experiment we observed that both anti-UmaA antibody and S-

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adenosyl-L-homocysteine, a non-methylated analog of SAM completely abrogated the

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methyl transfer. (Fig 2C, 2D, Lanes 2 and 3, respectively). Whereas, no change was

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observed in control reactions with crude extract of the E. coli containing empty vector

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pGEX-5x-3 (data not shown). All these observations corroborate specific action of

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UmaA on short chain fatty acids. To further validate the results, PcaA1, a recently

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characterized SAM dependent methyl transferase from M. tuberculosis (Glickman et al.,

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2000) was also used in the same reaction conditions as a reference enzyme. As

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expected, PcaA1 transferred majority of radiolabel to the MAMEs fraction (data not

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shown). These results collectively establish UmaA as a functional methyltransferase and

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identify short chain fatty acids as its potential substrate.

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To further gain insight into the nature of fatty acids modified by UmaA, we

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investigated its ability to modify artificial substrates in vitro. Previous studies hinted at a

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plausible involvment of a soluble enzyme from the extracts of Mycobacterium phlei in

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the enzymatic synthesis of short chain fatty acid, tuberculostearic acid (Akamatsu et al.,

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1970). The authors further concluded that TSA arises by direct methylation of

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phospholipid-linked oleic acid in the presence of S-adenosyl-L-methionine. These studies

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prompted us to investigate the phospholipid-linked oleic acid as a possible substrate of

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UmaA. This assumption was further supported by an observation that chemically

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synthesized methyl oleate migrated at an identical Rf value of 0.45 with the FAMEs

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fraction radiolabelled by UmaA (Fig 2A, Lane 7). In vitro reactions were carried out

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with a suitable phospholipid (L-a-phosphatidylcholine containing oleic acid at sn-2

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glycero position and saturated palmitic fatty acid at sn-1 position) and purified UmaA or

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E. coli crude lysate overexpressing UmaA in the presence of tritiated SAM. After

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saponification and methyl ester formation, the extracted radiolabeled product was

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analyzed with Bio-imaging Analyzer. Intriguingly, the radioactive spots obtained after an

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exposure of 96 hrs displayed identical Rf value when compared to the standard TSA

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methyl ester prepared separately (Fig 3A). This observation suggests that UmaA in the

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presence of SAM converts the olefinic bond of oleic acid into 10-methylstearic acid. The

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conversion of olefinic bond of oleic acid is a two step process. The chain is first

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alkenylated at the 10-carbon to give methylene group (10-methylene stearic acid) which

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is subsequently reduced to a stable methyl group (10-methyl stearic acid) by a hydrogen

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donor, NADPH. The addition of NADPH in the reaction would therefore drive the

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formation of a stable radiolabeled TSA. In the present study, a three-fold increase in

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label intensity was measured in the presence of NADPH (Fig 3B). To further corroborate

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the conversion of olefinic double bond of oleic acid to TSA, the extracted radiolabeled

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fatty acid fraction was subjected to oxidative periodate cleavage. Methyl oleate when

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used as a control was prone to periodate cleavage whereas the radiolabeled fatty acid

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fraction was non-susceptible to oxidative cleavage (Fig 4). These result established that

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the label was specifically incorporated at the double bond of oleic acid by UmaA.

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Therefore, these results put forward UmaA as the methyltransferase capable of

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converting olefinic bond of oleic acid to tuberculostearic acid in vitro. Results of present

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study is not in agreement with the UmaA mutants study of Laval et al. They observed

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that disruption of umaA in M. tuberculosis does not have any effect on composition of

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short chain fatty acids or mycolic acids (Laval et al., 2008). The possible reason for the

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discrepancy in the results could be due to the complex network of methyltransferases in

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M. tuberculosis wherein, function of one enzyme can be compensated by another

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enzyme. In our study we used purified UmaA enzyme and by employing various

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biochemical studies proved that UmaA is a methyltransferase capable of in vitro

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conversion of olefinic bond of oleic acid to tuberculostearic acid.

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Methyltransferases of mycobacteria were long proposed to be involved in the

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synthesis of methyl-branched short chain fatty acids, but the conception lacked direct

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experimental evidence (Campbell et al., 1969). The results presented in this study

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demonstrate UmaA of M. tuberculosis as a methytransferase capable of in vitro

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enzymatic modifications of short chain fatty acid. UmaA was shown to catalyze the

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conversion of phospholipid linked oleic acid to tuberculostearic acid in vitro.

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Tuberculostearic acid is a characteristic component of membrane lipids of mycobacteria

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(Ballou et al., 1963) and such a modification could imply a plausible adaptation to an

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environment encountered by bacterium where it encounters reactive oxygen species

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capable of degrading fatty acids by acting on olefinic bonds (Yuan et al., 1995). This

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hypothesis has been validated by an study in which McAdam et al, showed that M.

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tuberculosis carrying transposon in Rv0469 (umaA) is more virulent then wild type strain

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(McAdam et al., 2002). Thus, enzymes catalyzing such modifications offer viable target

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for chemotherapeutic intervention. Future work should focus on the examination of

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UmaA mutant to broaden our understanding on the role of UmaA in the survival and

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pathogenesis of M. tuberculosis.

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Materials
Biochemicals, chromatography materials, Freunds incomplete adjuvant and

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tuberculostearic acid (TSA) were purchased from Sigma (USA). The bacterial culture

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media and albumin dextrose complex (ADC) were obtained from Difco Laboratories

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(Becton Dickinson). Glutathione sepharose 4B resin, expression plasmid pGEX-5X-3

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and radiolabeled [3H]-SAM (84.0 Ci/mmol) were obtained from Amersham-Pharmacia.

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L--phoshpatidylcholine ([1-O-palmityl-2-O-oleicyl-sn-glycerol]-phoshpatidylcholine)

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and oleic acid was obtained from Arvanti Lipids and Merck, respectively. The pre-

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coated TLC plates (Silica Gel 60F254) were purchased from Merck and

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Trimethylorthoformate was obtained from Aldrich, sodium (Meta) periodate (Fluka),

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KMnO4 (Merck), NADPH (USL). The radioactivity on TLC plates was measured either

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on a scanner (Bioscan) or on a phosphoimager (Fujitsu). The liquid scintillation counter

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used was from Beckman (LS 5801) and Bio-imaging analyzer from Fujifilm FLA-5000.

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Bacterial culture and growth conditions

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MATERIALS AND METHODS

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M. tuberculosis strain H37Rv (obtained from Dr. J. S. Tyagi, AIIMS, New Delhi,

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India ) and M. smegmatis were grown in Middlebrook 7H9 broth supplemented with

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0.5% glycerol and 10% ADC at 37 C. E. coli strains DH5 and BL-21 were used for

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cloning and expression and were grown in LB broth or on LB agar plate at 37C.

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Plasmid construction

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M. tuberculosis H37Rv genomic DNA was used as a template for amplification of

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umaA by polymerase chain reaction (PCR) using the primers 5G AGA GGT TGG ATC

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CGC ATG ACT G 3 carrying BamH1 site (forward primer) and 5 G GGC GGC CTC

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GAG CTA CTT G 3 (reverse primer) carrying XhoI site. The PCR amplified fragment

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was digested with BamH1 and XhoI, and the resulting fragments were inserted into

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pGEX-5X-3 plasmid previously digested with same restriction enzymes.

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Expression and purification GST-UmaA

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GST-UmaA was affinity purified using glutathione-Sepharose-4B resin as

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described earlier (Meena et al., 2008 and Meena et al., 2012). In, brief the transformants

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were grown at 37 C under shaking until the Absorbance600 reached 0.6 and induced with

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1mM IPTG. Purified UmaA was used to raise polyclonal anti-UmaA antibody in rabbit.

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Localization of UmaA in mycobacterial cells

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Equal amount of protein (40 g each) from cell wall, cell membrane, cytoplasmic

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fractions and culture supernatant of M. tuberculosis were separated by 10 % SDS-PAGE.

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The proteins were electroblotted on a nitrocellulose membrane and probed with anti-

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UmaA serum raised in rabbit (1:1000 dilutions) in PBS containing 0.01% Tween-20.

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Anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary

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antibody and blot was developed using an ECL kit (Amersham-Pharmacia) according to

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manufacturers instructions.

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Cell-free assay for Methyl transferase activity


Crude cell lysate was prepared from 250 ml of M. smegmatis grown to an
Absorbance650 of 0.5-1.0. The cell pellet was washed twice with 25 ml of cold buffer

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(50mM Potassium phosphate, [pH-7.0], 1mM DTT, 1mM EDTA) and centrifuged at

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1200g at 40C for 10 min. The cells were re-suspended in 15 ml of cold buffer and lysed

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by sonication (40 second on/off, duty cycle 40%) for 15 minute. UmaA was over-

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expressed in E.coli BL-21-DE3 by growing transformed cells (carrying pGEX-umaA

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construct) in 250 ml of YT medium at 37 C and induced with 1mM IPTG at OD 0.5-0.6.

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The culture was grown for additional 4-5 hours at 37 C with shaking. At the end of

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incubation period cells were pelleted down, washed and lysed in GST sonication buffer at

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pH 7.4. Equal volume of substrate (M. smegmatis crude lysate) and protein (E. coli cell

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lysate) were mixed in a glass vial and incubated with 2.5 Ci [3H] S-adenosyl-L-

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methionine (250 Ci of 84.00 Ci/mmol) at 37C for one hour. The lipids were saponified

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overnight with equal volume of 15% tetrabutylammonium hydroxide (TBAH) at 800C.

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Samples were mixed with doubled volume of Dichloro methane, 4-5% Idomethane and

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incubated at room temperature for 2 hours. The upper aqueous phase was discarded and

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the lower organic phase was washed with water, 0.1N HCl and again with water. The

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lipids were extracted with diethyl ether, dried and finally dissolved in DCM. An aliquot

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of the resultant mixture of fatty acid methyl esters (FAMES) and mycolic acid methyl

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esters (MAMES) was then subjected to TLC plate and developed in petroleum ether/ether

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(9:1).

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Enzymatic activity of UmaA with L-


-Phosphatidyl Choline (PC)
L--phosphatidylcholine (1 mg/ml) containing saturated fatty acid (palmitic acid)

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at sn-1 position and an unsaturated (oleic acid) at sn-2 position was dispersed in 50 mM

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phosphate buffer (pH-8.0) containing 1mM EDTA and 1mM DTT. Equal volume of L-

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-PC (50 g) and E. coli lysate over-expressing UmaA and 1mM NADPH were mixed

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and incubated with 2.5 Ci of [3H]-SAM (250 Ci of 84.00 Ci/mmol) at 37 C for 1hr.

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The reaction was stopped with the addition of 6N HCl (2%). The lipids were saponified

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with equal volume of 25% KOH in methanol:water (1:1) at 1000C for 3-4 hours. After

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completion of the saponification, the reaction mixture was neutralized with acid (HCl:

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H2O, 1:1, 25%v/v) and free fatty acids were extracted with diethylether. Further, one ml

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of methanol:toluene:sulfuric-acid (30:15:1) and 5%v/v (trimethylorthoformate) was used

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for the preparation of the methyl esters. The mixture was incubated overnight at room

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temperature and the products were extracted into n-hexane. Finally, methyl esters were

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dissolved in DCM. Samples were applied on to the silica-gel TLC plate and finally

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developed using petroleum-ether:diethylether (9:1) solvent system.

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Chemical synthesis of Methyl Oleate and Oxidative Periodate test


Oleic acid was methyl esterified by using the MTS reagent (Metahnol: Toluene:

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Sulfuric acid, 30:15:1 and 5% trimethylorthoformate). The mixture was overnight

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incubated at room temperature. Oxidative periodate test-involved addition of tert-Butyl

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alcohol and 1ml of periodate reagent (Periodate: KMnO4, 39:1 w/w) followed by

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overnight incubation at room temperature. Finally, the methyl esters were extracted with

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hexane.

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ACKNOWLEDGEMENTS

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We thank Dr. Rajesh S. Gokhale, Director, CSIR-Institute of Genomics and

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Integrative Biology (IGIB), New Delhi, for making this work possible. One of the

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authors (LSM) wants to thanks the DST (Department of Science and Technology) for

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their financial support under the, DST grant numbers (GAP0050 and GAP0092) and

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the CSIR for providing funds under the In House Project Scheme (LSM59). Financial

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support was provided NMITLI, CSIR is also acknowledged. PC was supported by the

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university grant commission, Delhi, India.

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References

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Akamatsu, Y., and Law, J.H. (1970). Enzymatic alkylenation of phospholipid fatty acid

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mechanics of the mycobacterial cell wall: from horizontal layers to vertical scaffolds. J.

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Glickman, M.S., Cahill, S.M., and Jacobs Jr, W.R. (2000). The Mycobacterium

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tuberculosis cmaA2 gene encodes a mycolic acid trans-cyclopropane synthetase. J. Biol.

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Chem. 276, 2228-33.

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Goren, M. B. (1984). The mycobacteria: A sourcebook, Marcel Dekker, Inc., pp. 379-

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Grzegorzewicz, A.E., Kordulakova, J., Jones, V., Born, S.E., Belardinelli, J.M., Vaquie,

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A., Gundi, V.A., Madacki, J., Slama, N., Laval, F., Vaubourgeix, J., Crew, R.M.,

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Gicquel, B., Daffe, M., Morbidoni, H.R., Brennan, P.J., Quemard, A., McNeil, M.R., and

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Jackson, M. (2012). A Common Mechanism of Inhibition of the Mycobacterium

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tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone. J. Biol.

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Chem. 287, 38434-38441.

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Jackson, M., Crick, D.C., and Brennan, P.J. (2000). Phosphatidylinositol is an essential

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phospholipid of mycobacteria. J. Biol. Chem. 275, 30092-30099.

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Laval, F., Haites, R., Movahedzadeh, F., Lemassu, A., Wong, C.Y., Stoker, N., Billman-

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Jacobe, H., and Daffe, M. (2008). Investigating the function of the putative mycolic acid

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methyltransferase UmaA: divergence between the Mycobacterium smegmatis and

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Mycobacterium tuberculosis proteins. J. Biol. Chem. 283, 1419-1427.

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McAdam, R.A., Quan, S., Smith, D.A., Bardarov, S., Betts, J.C., Cook, F.C., Hooker,

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E.U., Lewis, A.P., Woollard, P., Everett, M.J., Lukey, P.T., Bancroft, G.J., Jacobs, Jr,

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WR, Jr., and Duncan, K. (2002). Characterization of a Mycobacterium tuberculosis

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H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered

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virulence. Microbiology. 148, 2975-2986.

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Meena, L.S., Chopra, P., Bedwal, R.S., and Singh, Y. (2008). Cloning and

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Characterization of a GTP binding protein from M. tuberculosis H37Rv. Enzyme. Microb.

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Meena, L.S., Dhakate, S. R., and Sahare, P.D. (2012). Elucidation of Mg2+ binding

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activity of adenylate kinase from Mycobacterium tuberculosis H37Rv using fluorescence

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studies. Biotechnol. Appl. Biochem. 59, 429-436.

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Nigou, J., Gilleron, M., Cahuzac, B., Bounery, J.D., Herold, M., Thurnher, M., and Puzo,

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G. (1997). The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from

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Mycobacterium bovis bacillus Calmette Guerin. Heterogeneity, structure, and role in the

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regulation of cytokine secretion. J. Biol. Chem. 272, 23094-23103.

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Slayden, R.A., DeBarber, A.E., Barry 3rd, C.E., Baird, M.S., Crick, D.C., and Brennan,

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P.J. (2003). Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium

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tuberculosis. J. Biol. Chem. 278, 53123-53130.

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Takayama, K., Wang, C., and Besra, G.S. (2005). Pathway to synthesis and processing

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of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18, 81-101.

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Yuan, Y., Lee, R.E., and Besra, G.S., Belisle, J.T., and Barry 3rd, C.E. (1995).

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Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in

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Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 92, 6630-6634.

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Yuan, Y., Mead, D., Schroeder, B.G., Zhu, Y., and Barry 3rd, C.E. (1998). The

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biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene)

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transfer to acyl carrier protein bound meromycolic acid in vitro. J. Biol. Chem. 273,

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21282-21290.

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FIGURE LEGENDS
Figure 1

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(A) Expression and purification of UmaA in E. coli

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E. coli cells harbouring pGEX-UmaA were grown in LB medium and induced with 1mM

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IPTG. Total lysates of E. coli expressing fusion protein was purified to homogeneity

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using GST beads. Lane 1, Molecular weight marker; Lane 2, GST-UmaA.

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(B) Sub cellular localization of UmaA in M. tuberculosis

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40 g protein each from cell wall, cell membrane, cytoplasm and whole cell lysate of M.

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tuberculosis were resolved by 10% SDS-PAGE and electroblotted on to nitrocellulose

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membrane. The blots were probed with anti-UmaA serum and developed using ECL

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reagents. Lane 1, Cytoplasmic fraction; Lane 2, Cell wall fraction; Lane 3, Cell

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membrane fraction; Lane 4, Whole cell lysates.

Expression, purification and sub cellular localization of UmaA

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Figure 2

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Biochemical characterization of UmaA

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(A) Cell free assay was performed using non heat treated (NHT) M. smegmatis crude cell

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lysates as a substrate, E. coli over expressing UmaA (E. coli-UmaA) as a source of

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enzyme and 2.5Ci of 84.00 Ci/mmol [3H] SAM as methyl group donor. Samples were

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resolved by Thin layer chromatography (Petroleum ether: Ether, 9:1).

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Lane 1, NHT + [3H] SAM, 7150cpm (Total loaded count); Lane 2, NHT + [3H] SAM +

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E. coli-UmaA, 44600cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ Anti-UmaA

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antibody,10600cpm; Lane 4, NHT + [3H] SAM + E. coli-UmaA+ S-Adenosyl-L-

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homocysteine (SAH), 4050cpm; Lane 5, Extracted and purified MAMES and FAMES;

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Lane 6, Purified MAMES; Lane 7, Chemically synthesized methyl oleate.

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(B) Assay using M. smegmatis crude lysate heat treated (HT) at 90 C for 10 min. Lane

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1, HT + [3H] SAM, 3160cpm; Lane 2, HT + [3H] SAM + GST-UmaA, 28100cpm; Lane

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3, HT + [3H] SAM + GST-UmaA + Anti-UmaA antibody, 10600cpm; Lane 4, HT + [3H]

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SAM + GST-UmaA + SAH, 2680cpm; Lane 5, Chemically synthesized methyl oleate;

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Lane 6, Extracted and purified MAMES and FAMES.

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(C) Assay in the presence of purified GST-UmaA. Lane 1, NHT + [3H] SAM + GST-

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UmaA, 47520cpm; Lane 2, NHT + [3H] SAM + GST-UmaA+ Anti-UmaA antibody,

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5000cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ SAH, 2720cpm; Lane 4,

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Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and

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FAMES

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(D) Assay in the presence of purified GST-UmaA. Lane 1, HT + [3H] SAM + GST-

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UmaA, 39520cpm; Lane 2, HT + [3H] SAM + GST-UmaA+ Anti-UmaA antibody,

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4120cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ SAH, 2720cpm; Lane 4,

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Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and

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FAMES

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Figure 3

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Methltransferase assay using Oleic acid linked to phopholipid (oleyl-PL) as an in

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vitro substrate.

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(A) Assay with E. coli over expressing UmaA and purified GST-UmaA as a source of

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enzyme in the presence of 2.5mCi of [3H]-SAM and oleyl-PL. Incorporation of

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radiolabel SAM is shown. Lane 2, 14720cpm; Lane 3, 11880 cpm

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(B) Oleyl-PC assay in the presence of 1mM NADPH and 2.5 mCi of [3H]-SAM and

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periodate cleavage test showing the formation of tuberculostearic acid. Enhanced

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radiolabeling is visualized in Lane 3 & 4. Lane 3, 38080 cpm; Lane 4, 35760cpm.

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Figure 4

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Non-susceptibility of the radiolabeled product to periodate cleavage

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The radiolabeled product formed under different reaction conditions was non-susceptible

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to periodate cleavage. Methyl oleates used as a control is cleaved on treatment with

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periodate.

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Figure 1

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116 kDa
97.4 kDa

66 kDa
47 kDa

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GSTUmaA1

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Figure 2
B

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FAMES

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MAMES

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Figure 3

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Metyl oleate

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Tuberculostearic
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SAH

Oleyl-PC + GST-UmaA1+NADPH
[methyl-3H] SAM
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Figure 4

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Tuberculostearic
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Tuberculostearic
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http://mc.manuscriptcentral.com/bc

Tuberculostearic
acid