Editing Manuscript of LSM

For Review Only

Biochemical characterization of an S-adenosyl-L-methionine

dependent methyltransferase (Rv0469) of Mycobacterium

tuberculosis

Journal: Biological Chemistry

Manuscript ID: BIOLCHEM-2013-0126

Manuscript Type: Research Article

Date Submitted by the Author: 23-Jan-2013

Complete List of Authors: Meena, Laxman; CSIR-Institute of Genomics and Integrative Biology,

Allergy and Infectious diseases Chopra, Puneet Vishwakarma, Ram Singh, Yogendra

Section/Category: Membranes, Lipids, Glycobiology

Keywords: Tuberculostearic-acid, S-adenosyl-L-methionine, Mycobacterium tuberculosis, Oleic-acid, methyltransferase, Fatty-acid

http://mc.manuscriptcentral.com/bc

Biological Chemistry

For Review Only

1

Biochemical characterization of an S-adenosyl-L-methionine dependent 1

methyltransferase (Rv0469) of Mycobacterium tuberculosis 2

3

4

Laxman S. Meena*, Puneet Chopra, Ram A. Vishwakarma and Yogendra Singh 5

6 CSIR-Institute of Genomics and Integrative Biology, Council of Scientific and 7

Industrial Research, Mall Road, Delhi-110007, and 8

Bio-organic Chemistry Lab, National Institute of Immunology, New Delhi 9

10

11

12

Running Title: Biosynthesis of Tuberculostearic acid 13

14 15 16 17 18 19 *To whom reprint request should be addressed: 20 21 * Dr. Laxman S. Meena, Ph.D 22 CSIR-Institute of Genomics and Integrative Biology 23 Mall Road, Delhi-110007 24 Telephone no: 011-27666156 25 Fax No: 011-27667471 26 E-mail ID: [email protected] 27 [email protected] 28 29

30

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Abstract 31

32 Tuberculostearic acid (l0-methylstearic acid, TSA) is a major constituent of 33

mycobacterial membrane phospholipids and its biosynthesis involves direct methylation 34

of oleic acid esterified as a component of phospholipids. Methyltransferases of 35

mycobacteria were long proposed to be involved in the synthesis of methyl-branched 36

short chain fatty acids, but direct experimental evidence is still lacking. In this study, we 37

identified methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a 38

novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase capable of 39

catalyzing the conversion of olefinic double bond of phospholipid linked oleic acid to 40

biologically essential tuberculostearic acid. Therefore UmaA catalyzing such 41

modifications offer viable target for chemotherapeutic intervention. 42

43

44

Key Words: Fatty-acid, S-adenosyl-L-methionine, Mycobacterium tuberculosis, 45

methyltransferase, Tuberculostearic-acid, Oleic-acid, 46

47 Abbreviations Used: PC, 1, 2 Dioleoyl-sn-Glycerol-3-phosphocholine; PE, 1, 2 48

Dioleoyl-sn-Glycerol-3-phosphoethanolamine; PS, 1, 2 Dioleoyl-sn-Glycerol-3-49

phosphoserine; SAM, S-adenosyl-L-methionine; TSA, Tuberculostearic acid 50

51 52 53 54 55

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Introduction 56 57

58 An important key to the success of pathogenic mycobacteria is its unusual cell 59

wall architecture. The characteristic cell wall core is composed of arabinogalactan-60

mycolate layer covalently linked to the cell wall peptidoglycan (Dmitriev et al., 2000), 61

phosphatidylinositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan 62

(LAM). The glycolipids derived metabolically from phosphatidylinositol (PI) are the 63

prominent interspersed phospholipids/lipoglycans of mycobacterial cell wall (Besra et al., 64

1997). They remain non-covalently attached to the plasma membrane through their 65

phosphatidyl myo-inositol anchor. Together, this highly complex array of lipids and 66

glycolipids form a thick barrier and protect mycobacterium from noxious chemicals as 67

well as during host infection. Therefore, the enzymes involved in the biosynthesis of this 68

essential structural component of M. tuberculosis H37Rv (Mycobacterium tuberculosis 69

H37Rv) offer a potential target for the chemotherapeutic intervention. 70

71

Among the potentially attractive drug targets are the enzymes involved in the 72

synthesis of the main mycobacterial phospholipids (Goren et al., 1984). 73

Phosphotidylinositol (PI) is an essential phospholipid of mycobacteria (Jackson et al., 74

2000) as it constitutes a lipid anchor to the cell envelop for PIMs, LM and LAM. The sn-75

1 and sn-2 positions of PI are acylated by C-16 and C-19 fatty acids respectively (Nigou 76

et al., 1997). The fatty acid at sn-2 position represents C-19 monomethyl-branched 77

stearic acid, Tuberculostearic acid (TSA). Tuberculostearic acid arises by methylation of 78

oleic acid esterified to phospholipids (oleyl-PL), with S-adenosylmethionine (SAM) as 79

the methyl donor. Oleic acid is first alkylenated at C-10 position to give 10-methylene 80

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stearic acid, which is subsequently reduced to 10-methylstearic acids with NADPH as a 81

cofactor (Akamatsu et al., 1970). Phetsuksiri et al., in their work on thiourea isoxyl 82

(ISO), a frontline anti-tuberculosis drug, identified the synthesis of oleic acid as the 83

primary target of ISO (Phetsuksiri et al., 2003). The authors also observed a dramatic 84

effect of ISO on the synthesis of tuberculostearic acid as a consequence of its effect on 85

oleic acid synthesis. The formation of TSA bears a strong resemblance to the enzymatic 86

modification of mycolic acids in M. tuberculosis. The double bonds in their 87

meromycolate chain are modified with cycopropane rings and methyl branches through 88

the action of a large family of SAM dependent methyltransferases (Takayama et al., 89

2005). Previous studies have established these highly homologous methyltransferases to 90

be functionally distinct. In a study, Grzegorzewicz et al., demonstrated that treatment of 91

M. tuberculosis with Isoxyl (ISO) and thiacetazone (TAC) inhibit the dehydratase step of 92

the fatty-acid synthase type II elongation cycle (Grzegorzewicz et al., 2012). Two 93

additional methyltransferases were also identified as the members of SAM dependent 94

methyl transferases family and were annotated as umaA and umaA2 (Cole et al., 1998). 95

Whereas umaA2 (PcaA1) was later characterized as a cyclopropane synthase, however, 96

umaA2 has not been biochemically characterized and its function is still not clear. 97

However, in a studs, Laval et al., showed that disruption of umaA in M. tuberculosis does 98

not have any effect on composition of short chain fatty acids or mycolic acids (Laval et 99

al., 2008). 100

101

In the present study we cloned, expressed and purified UmaA and investigated its 102

function and established it as a SAM dependent methyltransferase responsible for the 103

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modifications of short chain fatty acids. We demonstrate that UmaA is capable of 104

catalyzing the conversion of oleyl-PL to tuberculostearic acid in vitro. Thus UmaA 105

represents a family of methyltransferases involved in the biosynthesis of branched-short 106

chain fatty acids. 107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

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Results and Discussion 126

127 Methyltransferases of M. tuberculosis represent a large family of highly 128

homologous proteins involved in enzymatic modification of mycolic acids. The double 129

bonds in meromycolate chain of mycolic acids are catalyzed to cycolpropane ring and 130

methyl branches by the addition of methyl group derived from SAM. umaA shares 131

striking amino sequence similarity with the members of this gene family. Therefore, it 132

was pertinent to see whether umaA encodes a functional methyltransferase. In this study, 133

umaA gene was cloned in an E. coli expression vector, pGEX-5X-3. Over expression of 134

this protein resulted in a fusion protein of appropriate molecular weight of 59 kDa (33 135

kDa UmaA + 26 kDa GST tag) on a 10% SDS gel. Produced protein (UmaA) was 136

purified to homogeneity as GST fusion protein (Fig 1A). 137

The hydropathy profile predicted UmaA as a soluble protein. To confirm 138

bioinformatics prediction, sub cellular fractions of M. tuberculosis was prepared by 139

ultracentrifugation and purity of each sample was determined by checking specific 140

markers of that particular cellular fraction. Sub cellular fractions were separated by SDS-141

PAGE and western blotting was done using UmaA antisera. UmaA was detected as a 142

33-kDa protein in the whole cell lysate and cytoplasmic fractions and was absent from 143

both cell wall and cell membrane fractions (Fig 1B). These results confirmed that UmaA 144

is a cytoplasmic protein of M. tuberculosis H37Rv. 145

To biochemically characterize UmaA as a methyltransferase, a standard protocol 146

was followed (Yuan et al., 1998) to determine the optimal in vitro conditions. An initial 147

assay was performed with M. smegmatis crude lysate in the presence of radiolabelled 148

[methyl-3H] SAM as methyl group donor. After saponification and methyl ester 149

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preparation, the extracted products were analyzed on a silica TLC plate. In this study, the 150

majority of the label was transferred to fatty acid methyl ester (FAME) fractions, 151

representing short chain fatty acids. However, traces of radiolabel was also observed in 152

the mycolic acid methyl esters (MAMEs) fraction, representing the long chain fatty 153

acids (Fig 2A, Lane 1). To determine the specific activity of purified UmaA, the activity 154

of endogenous enzymes of M. smegmatis was eliminated by heat inactivation of crude 155

lysate at 90 for 10 min (Fig 2B, Lane 1). Under similar reaction conditions both heat 156

treated (HT) and non heat treated (NHT) M. smegmatis crude lysates were incubated with 157

crude extracts of E. coli cells overexpressing UmaA or purified UmaA in the presence of 158

radiolabelled SAM. In parallel control reactions with crude extract of the strain of E. coli 159

containing empty vector pGEX-5x-3 was also performed. Interestingly, the specific 160

labeling of FAMEs in HT (Fig 2B, Lane 2) and a substantial increase of radiolabel in 161

FAMEs fraction of NHT samples (Fig 2A, Lane 2) were observed with E. coli cells over-162

expressing UmaA. In the same study, we also observed that both anti-UmaA antibody 163

and S-adenosyl-L-homocysteine, a non-methylated analog of SAM completely abrogated 164

the methyl transfer. (Fig 2A, 2B, Lanes 3 and 4, respectively). Under similar condition, 165

purified UmaA protein also resulted in the specific labeling of FAMEs in HT Fig 2C, 166

Lane 1 and a substantial increase of radiolabel in FAMEs fraction of NHT samples (Fig 167

2D, Lane 1). In the same experiment we observed that both anti-UmaA antibody and S-168

adenosyl-L-homocysteine, a non-methylated analog of SAM completely abrogated the 169

methyl transfer. (Fig 2C, 2D, Lanes 2 and 3, respectively). Whereas, no change was 170

observed in control reactions with crude extract of the E. coli containing empty vector 171

pGEX-5x-3 (data not shown). All these observations corroborate specific action of 172

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UmaA on short chain fatty acids. To further validate the results, PcaA1, a recently 173

characterized SAM dependent methyl transferase from M. tuberculosis (Glickman et al., 174

2000) was also used in the same reaction conditions as a reference enzyme. As 175

expected, PcaA1 transferred majority of radiolabel to the MAMEs fraction (data not 176

shown). These results collectively establish UmaA as a functional methyltransferase and 177

identify short chain fatty acids as its potential substrate. 178

To further gain insight into the nature of fatty acids modified by UmaA, we 179

investigated its ability to modify artificial substrates in vitro. Previous studies hinted at a 180

plausible involvment of a soluble enzyme from the extracts of Mycobacterium phlei in 181

the enzymatic synthesis of short chain fatty acid, tuberculostearic acid (Akamatsu et al., 182

1970). The authors further concluded that TSA arises by direct methylation of 183

phospholipid-linked oleic acid in the presence of S-adenosyl-L-methionine. These studies 184

prompted us to investigate the phospholipid-linked oleic acid as a possible substrate of 185

UmaA. This assumption was further supported by an observation that chemically 186

synthesized methyl oleate migrated at an identical Rf value of 0.45 with the FAMEs 187

fraction radiolabelled by UmaA (Fig 2A, Lane 7). In vitro reactions were carried out 188

with a suitable phospholipid (L-a-phosphatidylcholine containing oleic acid at sn-2 189

glycero position and saturated palmitic fatty acid at sn-1 position) and purified UmaA or 190

E. coli crude lysate overexpressing UmaA in the presence of tritiated SAM. After 191

saponification and methyl ester formation, the extracted radiolabeled product was 192

analyzed with Bio-imaging Analyzer. Intriguingly, the radioactive spots obtained after an 193

exposure of 96 hrs displayed identical Rf value when compared to the standard TSA 194

methyl ester prepared separately (Fig 3A). This observation suggests that UmaA in the 195

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presence of SAM converts the olefinic bond of oleic acid into 10-methylstearic acid. The 196

conversion of olefinic bond of oleic acid is a two step process. The chain is first 197

alkenylated at the 10-carbon to give methylene group (10-methylene stearic acid) which 198

is subsequently reduced to a stable methyl group (10-methyl stearic acid) by a hydrogen 199

donor, NADPH. The addition of NADPH in the reaction would therefore drive the 200

formation of a stable radiolabeled TSA. In the present study, a three-fold increase in 201

label intensity was measured in the presence of NADPH (Fig 3B). To further corroborate 202

the conversion of olefinic double bond of oleic acid to TSA, the extracted radiolabeled 203

fatty acid fraction was subjected to oxidative periodate cleavage. Methyl oleate when 204

used as a control was prone to periodate cleavage whereas the radiolabeled fatty acid 205

fraction was non-susceptible to oxidative cleavage (Fig 4). These result established that 206

the label was specifically incorporated at the double bond of oleic acid by UmaA. 207

Therefore, these results put forward UmaA as the methyltransferase capable of 208

converting olefinic bond of oleic acid to tuberculostearic acid in vitro. Results of present 209

study is not in agreement with the UmaA mutants study of Laval et al. They observed 210

that disruption of umaA in M. tuberculosis does not have any effect on composition of 211

short chain fatty acids or mycolic acids (Laval et al., 2008). The possible reason for the 212

discrepancy in the results could be due to the complex network of methyltransferases in 213

M. tuberculosis wherein, function of one enzyme can be compensated by another 214

enzyme. In our study we used purified UmaA enzyme and by employing various 215

biochemical studies proved that UmaA is a methyltransferase capable of in vitro 216

conversion of olefinic bond of oleic acid to tuberculostearic acid. 217

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Methyltransferases of mycobacteria were long proposed to be involved in the 218

synthesis of methyl-branched short chain fatty acids, but the conception lacked direct 219

experimental evidence (Campbell et al., 1969). The results presented in this study 220

demonstrate UmaA of M. tuberculosis as a methytransferase capable of in vitro 221

enzymatic modifications of short chain fatty acid. UmaA was shown to catalyze the 222

conversion of phospholipid linked oleic acid to tuberculostearic acid in vitro. 223

Tuberculostearic acid is a characteristic component of membrane lipids of mycobacteria 224

(Ballou et al., 1963) and such a modification could imply a plausible adaptation to an 225

environment encountered by bacterium where it encounters reactive oxygen species 226

capable of degrading fatty acids by acting on olefinic bonds (Yuan et al., 1995). This 227

hypothesis has been validated by an study in which McAdam et al, showed that M. 228

tuberculosis carrying transposon in Rv0469 (umaA) is more virulent then wild type strain 229

(McAdam et al., 2002). Thus, enzymes catalyzing such modifications offer viable target 230

for chemotherapeutic intervention. Future work should focus on the examination of 231

UmaA mutant to broaden our understanding on the role of UmaA in the survival and 232

pathogenesis of M. tuberculosis. 233

234

235

236

237

238

239

240

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MATERIALS AND METHODS 241

Materials 242

Biochemicals, chromatography materials, Freunds incomplete adjuvant and 243

tuberculostearic acid (TSA) were purchased from Sigma (USA). The bacterial culture 244

media and albumin dextrose complex (ADC) were obtained from Difco Laboratories 245

(Becton Dickinson). Glutathione sepharose 4B resin, expression plasmid pGEX-5X-3 246

and radiolabeled [3H]-SAM (84.0 Ci/mmol) were obtained from Amersham-Pharmacia. 247

L--phoshpatidylcholine ([1-O-palmityl-2-O-oleicyl-sn-glycerol]-phoshpatidylcholine) 248

and oleic acid was obtained from Arvanti Lipids and Merck, respectively. The pre-249

coated TLC plates (Silica Gel 60F254) were purchased from Merck and 250

Trimethylorthoformate was obtained from Aldrich, sodium (Meta) periodate (Fluka), 251

KMnO4 (Merck), NADPH (USL). The radioactivity on TLC plates was measured either 252

on a scanner (Bioscan) or on a phosphoimager (Fujitsu). The liquid scintillation counter 253

used was from Beckman (LS 5801) and Bio-imaging analyzer from Fujifilm FLA-5000. 254

255

Bacterial culture and growth conditions 256

M. tuberculosis strain H37Rv (obtained from Dr. J. S. Tyagi, AIIMS, New Delhi, 257

India ) and M. smegmatis were grown in Middlebrook 7H9 broth supplemented with 258

0.5% glycerol and 10% ADC at 37 C. E. coli strains DH5 and BL-21 were used for 259

cloning and expression and were grown in LB broth or on LB agar plate at 37C. 260

Plasmid construction 261

M. tuberculosis H37Rv genomic DNA was used as a template for amplification of 262

umaA by polymerase chain reaction (PCR) using the primers 5G AGA GGT TGG ATC 263

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CGC ATG ACT G 3 carrying BamH1 site (forward primer) and 5 G GGC GGC CTC 264

GAG CTA CTT G 3 (reverse primer) carrying XhoI site. The PCR amplified fragment 265

was digested with BamH1 and XhoI, and the resulting fragments were inserted into 266

pGEX-5X-3 plasmid previously digested with same restriction enzymes. 267

268

Expression and purification GST-UmaA 269

GST-UmaA was affinity purified using glutathione-Sepharose-4B resin as 270

described earlier (Meena et al., 2008 and Meena et al., 2012). In, brief the transformants 271

were grown at 37 C under shaking until the Absorbance600 reached 0.6 and induced with 272

1mM IPTG. Purified UmaA was used to raise polyclonal anti-UmaA antibody in rabbit. 273

274

Localization of UmaA in mycobacterial cells 275

Equal amount of protein (40 g each) from cell wall, cell membrane, cytoplasmic 276

fractions and culture supernatant of M. tuberculosis were separated by 10 % SDS-PAGE. 277

The proteins were electroblotted on a nitrocellulose membrane and probed with anti-278

UmaA serum raised in rabbit (1:1000 dilutions) in PBS containing 0.01% Tween-20. 279

Anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary 280

antibody and blot was developed using an ECL kit (Amersham-Pharmacia) according to 281

manufacturers instructions. 282

283

Cell-free assay for Methyl transferase activity 284

Crude cell lysate was prepared from 250 ml of M. smegmatis grown to an 285

Absorbance650 of 0.5-1.0. The cell pellet was washed twice with 25 ml of cold buffer 286

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(50mM Potassium phosphate, [pH-7.0], 1mM DTT, 1mM EDTA) and centrifuged at 287

1200g at 40C for 10 min. The cells were re-suspended in 15 ml of cold buffer and lysed 288

by sonication (40 second on/off, duty cycle 40%) for 15 minute. UmaA was over-289

expressed in E.coli BL-21-DE3 by growing transformed cells (carrying pGEX-umaA 290

construct) in 250 ml of YT medium at 37 C and induced with 1mM IPTG at OD 0.5-0.6. 291

The culture was grown for additional 4-5 hours at 37 C with shaking. At the end of 292

incubation period cells were pelleted down, washed and lysed in GST sonication buffer at 293

pH 7.4. Equal volume of substrate (M. smegmatis crude lysate) and protein (E. coli cell 294

lysate) were mixed in a glass vial and incubated with 2.5 Ci [3H] S-adenosyl-L- 295

methionine (250 Ci of 84.00 Ci/mmol) at 37C for one hour. The lipids were saponified 296

overnight with equal volume of 15% tetrabutylammonium hydroxide (TBAH) at 800C. 297

Samples were mixed with doubled volume of Dichloro methane, 4-5% Idomethane and 298

incubated at room temperature for 2 hours. The upper aqueous phase was discarded and 299

the lower organic phase was washed with water, 0.1N HCl and again with water. The 300

lipids were extracted with diethyl ether, dried and finally dissolved in DCM. An aliquot 301

of the resultant mixture of fatty acid methyl esters (FAMES) and mycolic acid methyl 302

esters (MAMES) was then subjected to TLC plate and developed in petroleum ether/ether 303

(9:1). 304

305

Enzymatic activity of UmaA with L--Phosphatidyl Choline (PC) 306

L--phosphatidylcholine (1 mg/ml) containing saturated fatty acid (palmitic acid) 307

at sn-1 position and an unsaturated (oleic acid) at sn-2 position was dispersed in 50 mM 308

phosphate buffer (pH-8.0) containing 1mM EDTA and 1mM DTT. Equal volume of L-309

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-PC (50 g) and E. coli lysate over-expressing UmaA and 1mM NADPH were mixed 310

and incubated with 2.5 Ci of [3H]-SAM (250 Ci of 84.00 Ci/mmol) at 37 C for 1hr. 311

The reaction was stopped with the addition of 6N HCl (2%). The lipids were saponified 312

with equal volume of 25% KOH in methanol:water (1:1) at 1000C for 3-4 hours. After 313

completion of the saponification, the reaction mixture was neutralized with acid (HCl: 314

H2O, 1:1, 25%v/v) and free fatty acids were extracted with diethylether. Further, one ml 315

of methanol:toluene:sulfuric-acid (30:15:1) and 5%v/v (trimethylorthoformate) was used 316

for the preparation of the methyl esters. The mixture was incubated overnight at room 317

temperature and the products were extracted into n-hexane. Finally, methyl esters were 318

dissolved in DCM. Samples were applied on to the silica-gel TLC plate and finally 319

developed using petroleum-ether:diethylether (9:1) solvent system. 320

321

Chemical synthesis of Methyl Oleate and Oxidative Periodate test 322

Oleic acid was methyl esterified by using the MTS reagent (Metahnol: Toluene: 323

Sulfuric acid, 30:15:1 and 5% trimethylorthoformate). The mixture was overnight 324

incubated at room temperature. Oxidative periodate test-involved addition of tert-Butyl 325

alcohol and 1ml of periodate reagent (Periodate: KMnO4, 39:1 w/w) followed by 326

overnight incubation at room temperature. Finally, the methyl esters were extracted with 327

hexane. 328

329

330

331

332

333

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ACKNOWLEDGEMENTS 334

335

We thank Dr. Rajesh S. Gokhale, Director, CSIR-Institute of Genomics and 336

Integrative Biology (IGIB), New Delhi, for making this work possible. One of the 337

authors (LSM) wants to thanks the DST (Department of Science and Technology) for 338

their financial support under the, DST grant numbers (GAP0050 and GAP0092) and 339

the CSIR for providing funds under the In House Project Scheme (LSM59). Financial 340

support was provided NMITLI, CSIR is also acknowledged. PC was supported by the 341

university grant commission, Delhi, India. 342

343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369

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References 370 371 372 Akamatsu, Y., and Law, J.H. (1970). Enzymatic alkylenation of phospholipid fatty acid 373

chains by extracts of Mycobacterium phlei. J. Biol. Chem. 245, 701-708. 374

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Ballou, C.E., Vilkas, E., and Lederer, E. (1963). Structural studies on the myo-inositol 376

phospholipids of Mycobacterium tuberculosis (var. bovis, strain BCG. J. Biol. Chem. 377

238, 69-76. 378

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Besra, G.S., Morehouse, C.B., Rittner, C.M., Waechter, C.J., and Brennan, P.J. (1997). 380

Biosynthesis of mycobacterial lipoarabinomannan. J. Biol. Chem. 272, 18460-18466. 381

382

Campbell, I.M., and Naworal, J. (1969). Composition of the saturated and 383

monounsaturated fatty acids of Mycobacterium phlei. J. Lipid. Res. 10, 593-598. 384

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Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., 386

Eiglmeier, K., Gas, S., Barry, C.E., Tekaia, F., Badcock, K., Basham, D., Brown, D., 387

Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, 388

N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., 389

Oliver, K., Osborne, J., Quaol, M.A., Rajandream, M.A., Rogers, R., Rutter, S., Seeger, 390

K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S., and 391

Barrell, B.G. (1998). Deciphering the biology of Mycobacterium tuberculosis from the 392

complete genome sequence. Nature. 393, 537-544. 393

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Dmitriev, B.A., Ehlers, S., Rietschel, E.T., and Brennan, P.J. (2000). Molecular 395

mechanics of the mycobacterial cell wall: from horizontal layers to vertical scaffolds. J. 396

Med. Microbiol. 290, 251-258. 397

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Glickman, M.S., Cahill, S.M., and Jacobs Jr, W.R. (2000). The Mycobacterium 399

tuberculosis cmaA2 gene encodes a mycolic acid trans-cyclopropane synthetase. J. Biol. 400

Chem. 276, 2228-33. 401

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Goren, M. B. (1984). The mycobacteria: A sourcebook, Marcel Dekker, Inc., pp. 379-403

415. New York, 404

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Grzegorzewicz, A.E., Kordulakova, J., Jones, V., Born, S.E., Belardinelli, J.M., Vaquie, 406

A., Gundi, V.A., Madacki, J., Slama, N., Laval, F., Vaubourgeix, J., Crew, R.M., 407

Gicquel, B., Daffe, M., Morbidoni, H.R., Brennan, P.J., Quemard, A., McNeil, M.R., and 408

Jackson, M. (2012). A Common Mechanism of Inhibition of the Mycobacterium 409

tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone. J. Biol. 410

Chem. 287, 38434-38441. 411

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Jackson, M., Crick, D.C., and Brennan, P.J. (2000). Phosphatidylinositol is an essential 413

phospholipid of mycobacteria. J. Biol. Chem. 275, 30092-30099. 414

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Laval, F., Haites, R., Movahedzadeh, F., Lemassu, A., Wong, C.Y., Stoker, N., Billman-416

Jacobe, H., and Daffe, M. (2008). Investigating the function of the putative mycolic acid 417

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methyltransferase UmaA: divergence between the Mycobacterium smegmatis and 418

Mycobacterium tuberculosis proteins. J. Biol. Chem. 283, 1419-1427. 419

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McAdam, R.A., Quan, S., Smith, D.A., Bardarov, S., Betts, J.C., Cook, F.C., Hooker, 421

E.U., Lewis, A.P., Woollard, P., Everett, M.J., Lukey, P.T., Bancroft, G.J., Jacobs, Jr, 422

WR, Jr., and Duncan, K. (2002). Characterization of a Mycobacterium tuberculosis 423

H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered 424

virulence. Microbiology. 148, 2975-2986. 425

426

Meena, L.S., Chopra, P., Bedwal, R.S., and Singh, Y. (2008). Cloning and 427

Characterization of a GTP binding protein from M. tuberculosis H37Rv. Enzyme. Microb. 428

Technol. 42, 138-144. 429

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Meena, L.S., Dhakate, S. R., and Sahare, P.D. (2012). Elucidation of Mg2+ binding 431

activity of adenylate kinase from Mycobacterium tuberculosis H37Rv using fluorescence 432

studies. Biotechnol. Appl. Biochem. 59, 429-436. 433

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G. (1997). The phosphatidyl-myo-inositol anchor of the lipoarabinomannans from 436

Mycobacterium bovis bacillus Calmette Guerin. Heterogeneity, structure, and role in the 437

regulation of cytokine secretion. J. Biol. Chem. 272, 23094-23103. 438

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Phetsuksiri, B., Jackson, M., Scherman, H., McNeil, M., Besra, G.S., Baulard, A.R., 440

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P.J. (2003). Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium 442

tuberculosis. J. Biol. Chem. 278, 53123-53130. 443

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Takayama, K., Wang, C., and Besra, G.S. (2005). Pathway to synthesis and processing 445

of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18, 81-101. 446

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Yuan, Y., Lee, R.E., and Besra, G.S., Belisle, J.T., and Barry 3rd, C.E. (1995). 448

Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in 449

Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 92, 6630-6634. 450

451

Yuan, Y., Mead, D., Schroeder, B.G., Zhu, Y., and Barry 3rd, C.E. (1998). The 452

biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene) 453

transfer to acyl carrier protein bound meromycolic acid in vitro. J. Biol. Chem. 273, 454

21282-21290. 455

456 457 458

459

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460 FIGURE LEGENDS 461

Figure 1 462 463 Expression, purification and sub cellular localization of UmaA 464 465 (A) Expression and purification of UmaA in E. coli 466

E. coli cells harbouring pGEX-UmaA were grown in LB medium and induced with 1mM 467

IPTG. Total lysates of E. coli expressing fusion protein was purified to homogeneity 468

using GST beads. Lane 1, Molecular weight marker; Lane 2, GST-UmaA. 469

(B) Sub cellular localization of UmaA in M. tuberculosis 470

40 g protein each from cell wall, cell membrane, cytoplasm and whole cell lysate of M. 471

tuberculosis were resolved by 10% SDS-PAGE and electroblotted on to nitrocellulose 472

membrane. The blots were probed with anti-UmaA serum and developed using ECL 473

reagents. Lane 1, Cytoplasmic fraction; Lane 2, Cell wall fraction; Lane 3, Cell 474

membrane fraction; Lane 4, Whole cell lysates. 475

476

Figure 2 477

Biochemical characterization of UmaA 478

(A) Cell free assay was performed using non heat treated (NHT) M. smegmatis crude cell 479

lysates as a substrate, E. coli over expressing UmaA (E. coli-UmaA) as a source of 480

enzyme and 2.5Ci of 84.00 Ci/mmol [3H] SAM as methyl group donor. Samples were 481

resolved by Thin layer chromatography (Petroleum ether: Ether, 9:1). 482

Lane 1, NHT + [3H] SAM, 7150cpm (Total loaded count); Lane 2, NHT + [3H] SAM + 483

E. coli-UmaA, 44600cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ Anti-UmaA 484

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antibody,10600cpm; Lane 4, NHT + [3H] SAM + E. coli-UmaA+ S-Adenosyl-L-485

homocysteine (SAH), 4050cpm; Lane 5, Extracted and purified MAMES and FAMES; 486

Lane 6, Purified MAMES; Lane 7, Chemically synthesized methyl oleate. 487

488

(B) Assay using M. smegmatis crude lysate heat treated (HT) at 90 C for 10 min. Lane 489

1, HT + [3H] SAM, 3160cpm; Lane 2, HT + [3H] SAM + GST-UmaA, 28100cpm; Lane 490

3, HT + [3H] SAM + GST-UmaA + Anti-UmaA antibody, 10600cpm; Lane 4, HT + [3H] 491

SAM + GST-UmaA + SAH, 2680cpm; Lane 5, Chemically synthesized methyl oleate; 492

Lane 6, Extracted and purified MAMES and FAMES. 493

494

(C) Assay in the presence of purified GST-UmaA. Lane 1, NHT + [3H] SAM + GST-495

UmaA, 47520cpm; Lane 2, NHT + [3H] SAM + GST-UmaA+ Anti-UmaA antibody, 496

5000cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ SAH, 2720cpm; Lane 4, 497

Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and 498

FAMES 499

500

(D) Assay in the presence of purified GST-UmaA. Lane 1, HT + [3H] SAM + GST-501

UmaA, 39520cpm; Lane 2, HT + [3H] SAM + GST-UmaA+ Anti-UmaA antibody, 502

4120cpm; Lane 3, NHT + [3H] SAM + E. coli-UmaA+ SAH, 2720cpm; Lane 4, 503

Chemically synthesized methyl oleate; Lane 5, Extracted and purified MAMES and 504

FAMES 505

506

507

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Figure 3 508

Methltransferase assay using Oleic acid linked to phopholipid (oleyl-PL) as an in 509

vitro substrate. 510

(A) Assay with E. coli over expressing UmaA and purified GST-UmaA as a source of 511

enzyme in the presence of 2.5mCi of [3H]-SAM and oleyl-PL. Incorporation of 512

radiolabel SAM is shown. Lane 2, 14720cpm; Lane 3, 11880 cpm 513

514

(B) Oleyl-PC assay in the presence of 1mM NADPH and 2.5 mCi of [3H]-SAM and 515

periodate cleavage test showing the formation of tuberculostearic acid. Enhanced 516

radiolabeling is visualized in Lane 3 & 4. Lane 3, 38080 cpm; Lane 4, 35760cpm. 517

518

Figure 4 519

Non-susceptibility of the radiolabeled product to periodate cleavage 520

The radiolabeled product formed under different reaction conditions was non-susceptible 521

to periodate cleavage. Methyl oleates used as a control is cleaved on treatment with 522

periodate. 523

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Figure 1

97.4 kDa

GST- M UmaA1

116 kDa

66 kDa

47 kDa

31 kDa

A

UmaA1

B

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1 2 3 4 5 6

Figure 2

FAMES

1 2 3 4 5

C

FAMES

MAMES

FAMES

B

1 2 3 4 5 6 7

MAMES

Methyl oleate FAMES

A

FAMES

1 2 3 4 5

D

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Figure 3

Metyl oleate Radilabeled species

Meth

yl o

leate

Ole

yl-P

C +

E.coli

Um

aA

1+

[meth

yl-3

H]

SA

M

Ole

yl-P

C +

GS

T-U

maA

1+

[meth

yl-3

H]

SA

M

A

1 2 3

Tuberculostearic

acid

Syn

thetic T

SA

MA

ME

s +

FA

ME

s

Ole

yl-P

C +

E.coli

Um

aA

1+

NA

DP

H

[meth

yl-3

H]

SA

M

Ole

yl-P

C +

GS

T-U

maA

1+

NA

DP

H

[meth

yl-3

H]

SA

M

Anti-U

maA

1

SA

H

B

1 2 3 4 5 6

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Figure 4

A

Tuberculostearic

acid

C

Tuberculostearic

acid

B

Tuberculostearic

acid

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Editing Manuscript of LSM