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Research Article
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BIOLCHEM-2013-0126
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Biological Chemistry
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Biological Chemistry
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Abstract
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short chain fatty acids, but direct experimental evidence is still lacking. In this study, we
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catalyzing the conversion of olefinic double bond of phospholipid linked oleic acid to
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Biological Chemistry
Introduction
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mycolate layer covalently linked to the cell wall peptidoglycan (Dmitriev et al., 2000),
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(LAM). The glycolipids derived metabolically from phosphatidylinositol (PI) are the
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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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Among the potentially attractive drug targets are the enzymes involved in the
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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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the methyl donor. Oleic acid is first alkylenated at C-10 position to give 10-methylene
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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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oleic acid synthesis. The formation of TSA bears a strong resemblance to the enzymatic
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meromycolate chain are modified with cycopropane rings and methyl branches through
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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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methyl transferases family and were annotated as umaA and umaA2 (Cole et al., 1998).
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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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Biological Chemistry
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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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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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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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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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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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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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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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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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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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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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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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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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donor, NADPH. The addition of NADPH in the reaction would therefore drive the
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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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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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enzyme. In our study we used purified UmaA enzyme and by employing various
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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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enzymatic modifications of short chain fatty acid. UmaA was shown to catalyze the
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(Ballou et al., 1963) and such a modification could imply a plausible adaptation to an
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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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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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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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KMnO4 (Merck), NADPH (USL). The radioactivity on TLC plates was measured either
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used was from Beckman (LS 5801) and Bio-imaging analyzer from Fujifilm FLA-5000.
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Biological Chemistry
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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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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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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Equal amount of protein (40 g each) from cell wall, cell membrane, cytoplasmic
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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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antibody and blot was developed using an ECL kit (Amersham-Pharmacia) according to
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manufacturers instructions.
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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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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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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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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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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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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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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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References
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Akamatsu, Y., and Law, J.H. (1970). Enzymatic alkylenation of phospholipid fatty acid
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Ballou, C.E., Vilkas, E., and Lederer, E. (1963). Structural studies on the myo-inositol
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238, 69-76.
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r
Fo
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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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385
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On
386
Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V.,
387
Eiglmeier, K., Gas, S., Barry, C.E., Tekaia, F., Badcock, K., Basham, D., Brown, D.,
388
Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin,
389
N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L.,
390
Oliver, K., Osborne, J., Quaol, M.A., Rajandream, M.A., Rogers, R., Rutter, S., Seeger,
391
K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S., and
392
Barrell, B.G. (1998). Deciphering the biology of Mycobacterium tuberculosis from the
393
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Dmitriev, B.A., Ehlers, S., Rietschel, E.T., and Brennan, P.J. (2000). Molecular
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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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r
Fo
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Goren, M. B. (1984). The mycobacteria: A sourcebook, Marcel Dekker, Inc., pp. 379-
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vi
Re
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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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Jackson, M., Crick, D.C., and Brennan, P.J. (2000). Phosphatidylinositol is an essential
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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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H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered
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Fo
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Meena, L.S., Chopra, P., Bedwal, R.S., and Singh, Y. (2008). Cloning and
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ew
vi
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Re
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Meena, L.S., Dhakate, S. R., and Sahare, P.D. (2012). Elucidation of Mg2+ binding
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Mycobacterium bovis bacillus Calmette Guerin. Heterogeneity, structure, and role in the
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Phetsuksiri, B., Jackson, M., Scherman, H., McNeil, M., Besra, G.S., Baulard, A.R.,
441
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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Takayama, K., Wang, C., and Besra, G.S. (2005). Pathway to synthesis and processing
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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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Yuan, Y., Mead, D., Schroeder, B.G., Zhu, Y., and Barry 3rd, C.E. (1998). The
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transfer to acyl carrier protein bound meromycolic acid in vitro. J. Biol. Chem. 273,
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Figure 1
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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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40 g protein each from cell wall, cell membrane, cytoplasm and whole cell lysate of M.
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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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(A) Cell free assay was performed using non heat treated (NHT) M. smegmatis crude cell
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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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Lane 1, NHT + [3H] SAM, 7150cpm (Total loaded count); Lane 2, NHT + [3H] SAM +
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homocysteine (SAH), 4050cpm; Lane 5, Extracted and purified MAMES and FAMES;
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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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(C) Assay in the presence of purified GST-UmaA. Lane 1, NHT + [3H] SAM + GST-
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Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and
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Figure 3
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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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(B) Oleyl-PC assay in the presence of 1mM NADPH and 2.5 mCi of [3H]-SAM and
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Figure 4
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The radiolabeled product formed under different reaction conditions was non-susceptible
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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
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Figure 2
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A
FAMES
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MAMES
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Methyl oleate
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Figure 3
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Metyl oleate
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Radilabeled species
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SAH
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Figure 4
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Tuberculostearic
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