Editing Manuscript of LSM

Biological Chemistry
Biochemical characterization of an S-adenosyl-L-methionine

dependent methyltransferase (Rv0469) of Mycobacterium
tuberculosis
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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:
Tuberculostearic-acid, S-adenosyl-L-methionine, Mycobacterium

tuberculosis, Oleic-acid, methyltransferase, Fatty-acid
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http://mc.manuscriptcentral.com/bc
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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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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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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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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
289
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
291
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
293
incubation period cells were pelleted down, washed and lysed in GST sonication buffer at
294
pH 7.4. Equal volume of substrate (M. smegmatis crude lysate) and protein (E. coli cell
295
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
297
overnight with equal volume of 15% tetrabutylammonium hydroxide (TBAH) at 800C.
298
Samples were mixed with doubled volume of Dichloro methane, 4-5% Idomethane and
299
incubated at room temperature for 2 hours. The upper aqueous phase was discarded and
300
the lower organic phase was washed with water, 0.1N HCl and again with water. The
301
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
303
esters (MAMES) was then subjected to TLC plate and developed in petroleum ether/ether
304
(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
309
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.
312
The reaction was stopped with the addition of 6N HCl (2%). The lipids were saponified
313
with equal volume of 25% KOH in methanol:water (1:1) at 1000C for 3-4 hours. After
314
completion of the saponification, the reaction mixture was neutralized with acid (HCl:
315
H2O, 1:1, 25%v/v) and free fatty acids were extracted with diethylether. Further, one ml
316
of methanol:toluene:sulfuric-acid (30:15:1) and 5%v/v (trimethylorthoformate) was used
317
for the preparation of the methyl esters. The mixture was incubated overnight at room
318
temperature and the products were extracted into n-hexane. Finally, methyl esters were
319
dissolved in DCM. Samples were applied on to the silica-gel TLC plate and finally
320
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:
On
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Sulfuric acid, 30:15:1 and 5% trimethylorthoformate). The mixture was overnight
325
incubated at room temperature. Oxidative periodate test-involved addition of tert-Butyl
326
alcohol and 1ml of periodate reagent (Periodate: KMnO4, 39:1 w/w) followed by
327
overnight incubation at room temperature. Finally, the methyl esters were extracted with
328
hexane.
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ACKNOWLEDGEMENTS
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We thank Dr. Rajesh S. Gokhale, Director, CSIR-Institute of Genomics and
337
Integrative Biology (IGIB), New Delhi, for making this work possible. One of the
338
authors (LSM) wants to thanks the DST (Department of Science and Technology) for
339
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
341
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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Ballou, C.E., Vilkas, E., and Lederer, E. (1963). Structural studies on the myo-inositol
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Besra, G.S., Morehouse, C.B., Rittner, C.M., Waechter, C.J., and Brennan, P.J. (1997).
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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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Goren, M. B. (1984). The mycobacteria: A sourcebook, Marcel Dekker, Inc., pp. 379-
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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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Technol. 42, 138-144.
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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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Phetsuksiri, B., Jackson, M., Scherman, H., McNeil, M., Besra, G.S., Baulard, A.R.,
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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
446
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
467
E. coli cells harbouring pGEX-UmaA were grown in LB medium and induced with 1mM
468
IPTG. Total lysates of E. coli expressing fusion protein was purified to homogeneity
469
using GST beads. Lane 1, Molecular weight marker; Lane 2, GST-UmaA.
470
(B) Sub cellular localization of UmaA in M. tuberculosis
471
40 g protein each from cell wall, cell membrane, cytoplasm and whole cell lysate of M.
472
tuberculosis were resolved by 10% SDS-PAGE and electroblotted on to nitrocellulose
473
membrane. The blots were probed with anti-UmaA serum and developed using ECL
474
reagents. Lane 1, Cytoplasmic fraction; Lane 2, Cell wall fraction; Lane 3, Cell
475
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
479
(A) Cell free assay was performed using non heat treated (NHT) M. smegmatis crude cell
480
lysates as a substrate, E. coli over expressing UmaA (E. coli-UmaA) as a source of
481
enzyme and 2.5Ci of 84.00 Ci/mmol [3H] SAM as methyl group donor. Samples were
482
resolved by Thin layer chromatography (Petroleum ether: Ether, 9:1).
483
Lane 1, NHT + [3H] SAM, 7150cpm (Total loaded count); Lane 2, NHT + [3H] SAM +
484
E. coli-UmaA, 44600cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ Anti-UmaA
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485
antibody,10600cpm; Lane 4, NHT + [3H] SAM + E. coli-UmaA+ S-Adenosyl-L-
486
homocysteine (SAH), 4050cpm; Lane 5, Extracted and purified MAMES and FAMES;
487
Lane 6, Purified MAMES; Lane 7, Chemically synthesized methyl oleate.
488
489
(B) Assay using M. smegmatis crude lysate heat treated (HT) at 90 C for 10 min. Lane
490
1, HT + [3H] SAM, 3160cpm; Lane 2, HT + [3H] SAM + GST-UmaA, 28100cpm; Lane
491
3, HT + [3H] SAM + GST-UmaA + Anti-UmaA antibody, 10600cpm; Lane 4, HT + [3H]
492
SAM + GST-UmaA + SAH, 2680cpm; Lane 5, Chemically synthesized methyl oleate;
493
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-
496
UmaA, 47520cpm; Lane 2, NHT + [3H] SAM + GST-UmaA+ Anti-UmaA antibody,
497
5000cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ SAH, 2720cpm; Lane 4,
498
Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and
499
FAMES
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(D) Assay in the presence of purified GST-UmaA. Lane 1, HT + [3H] SAM + GST-
502
UmaA, 39520cpm; Lane 2, HT + [3H] SAM + GST-UmaA+ Anti-UmaA antibody,
503
4120cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ SAH, 2720cpm; Lane 4,
504
Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and
505
FAMES
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Figure 3
509
Methltransferase assay using Oleic acid linked to phopholipid (oleyl-PL) as an in
510
vitro substrate.
511
(A) Assay with E. coli over expressing UmaA and purified GST-UmaA as a source of
512
enzyme in the presence of 2.5mCi of [3H]-SAM and oleyl-PL. Incorporation of
513
radiolabel SAM is shown. Lane 2, 14720cpm; Lane 3, 11880 cpm
514
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(B) Oleyl-PC assay in the presence of 1mM NADPH and 2.5 mCi of [3H]-SAM and
516
periodate cleavage test showing the formation of tuberculostearic acid. Enhanced
517
radiolabeling is visualized in Lane 3 & 4. Lane 3, 38080 cpm; Lane 4, 35760cpm.
518
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Figure 4
520
Non-susceptibility of the radiolabeled product to periodate cleavage
521
The radiolabeled product formed under different reaction conditions was non-susceptible
522
to periodate cleavage. Methyl oleates used as a control is cleaved on treatment with
523
periodate.
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Figure 1
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116 kDa
97.4 kDa
66 kDa
47 kDa
31 kDa
GSTUmaA1
rR
ev
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UmaA1
On
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Figure 2
B
A
FAMES
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Page 24 of 26
MAMES
1 2
3 4
rR
6
MAMES
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D
FAMES
FAMES
Methyl oleate
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FAMES
FAMES
Page 25 of 26
Figure 3
Fo
2
rR
Metyl oleate
4 5
Tuberculostearic
acid
Radilabeled species
SAH
Oleyl-PC + GST-UmaA1+NADPH
[methyl-3H] SAM
Anti-UmaA1
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Oleyl-PC + E.coli UmaA1+ NADPH

[methyl-3H] SAM
On
MAMEs + FAMEs
iew
Synthetic TSA
ev
Methyl oleate
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Oleyl-PC + GST-UmaA1+
[methyl-3H] SAM
Oleyl-PC + E.coli UmaA1+

[methyl-3H] SAM
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Figure 4
Fo
rR
Tuberculostearic
acid
ev
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Tuberculostearic
acid
On
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Tuberculostearic
acid

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Biological Chemistry

Biochemical characterization of an S-adenosyl-L-methionine

Complete List of Authors:

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

Date Submitted by the Author:

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

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

methyltransferase (Rv0469) of Mycobacterium tuberculosis

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

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

Running Title: Biosynthesis of Tuberculostearic acid

*To whom reprint request should be addressed:

* Dr. Laxman S. Meena, Ph.D

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

mycobacterial membrane phospholipids and its biosynthesis involves direct methylation

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

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

identified methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a

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

biologically essential tuberculostearic acid. Therefore UmaA catalyzing such

modifications offer viable target for chemotherapeutic intervention.

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

methyltransferase, Tuberculostearic-acid, Oleic-acid,

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

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

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

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

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

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

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

essential structural component of M. tuberculosis H37Rv (Mycobacterium tuberculosis

H37Rv) offer a potential target for the chemotherapeutic intervention.

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

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

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

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

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

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

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

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

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

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

additional methyltransferases were also identified as the members of SAM dependent

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

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

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

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

chain fatty acids.

Results and Discussion

Methyltransferases of M. tuberculosis represent a large family of highly

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

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

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

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

ultracentrifugation and purity of each sample was determined by checking specific

is a cytoplasmic protein of M. tuberculosis H37Rv.

To biochemically characterize UmaA as a methyltransferase, a standard protocol

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

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

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

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

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