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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
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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
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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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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
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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
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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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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
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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
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methyltransferase UmaA: divergence between the Mycobacterium smegmatis and 418
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activity of adenylate kinase from Mycobacterium tuberculosis H37Rv using fluorescence 432
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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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of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18, 81-101. 446
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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
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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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