From the Tuberculosis Research Section, Laboratory of Intracellular
Parasites, Rocky Mountain Laboratories, NIAID, NIH,
Hamilton, Montana 59840
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INTRODUCTION |
Mycobacterium tuberculosis replicates within the
hostile environment of the mammalian macrophage whose microbicidal
products are generally insufficient to kill this highly resistant
bacillus. This intrinsic resistance to antibacterial substances is due, in large part, to the impermeable nature of the mycobacterial cell wall
(1-3). The mycobacterial cell wall consists of three covalently
attached polymers: the peptidoglycan, the arabinogalactan, and the
mycolic acids. The very low fluidity of the hydrophobic domain of this
structure significantly reduces the rate at which hydrophobic
substances are taken in and thereby potentiates the toxicity of such
substances. This low fluidity is directly attributable to the structure
of the mycolic acids, which comprise a large proportion of the cell
wall mass (4). Because of the essential nature of this structure to the
intracellular life of M. tuberculosis, its biosynthesis and
assembly offer critical potential targets for chemotherapeutic
intervention.
Mycolic acids are
-alkyl-
-hydroxy fatty acids of exceptional
length and complexity, which range up to 80 carbons in total chain
length and include various functional groups such as cis or
trans cyclopropanes or olefins,
-methyl methyl ethers, or
-methyl ketones. Because of their structural complexity and issues of overlap with enzymes responsible for synthesizing short-chain fatty
acids, in vitro systems for studying their biosynthesis have
been difficult to develop. We have identified a family of six
homologous enzymes by heterologous expression in the saprophytic mycobacterial species Mycobacterium smegmatis that are
involved in generating structural diversity among these molecules by
the apparent addition of a methyl group from
S-adenosyl-L-methionine (SAM)1 (5-8). The mechanism
of methylation by these enzymes has been proposed to involve the
formation of an intermediate carbocation, which can then react to form
a variety of different chemical structures depending upon the active
site configuration of the particular enzyme resulting in the observed
structural diversity of mycolic acids.
Although heterologous expression studies have proven useful for
identifying the genes encoding the enzymes involved in mycolic acid
modification, they have not resolved critical issues regarding the
substrate for methyl(ene) group transfer, including lipid chain length
during SAM addition and the head group or acyl carrier moiety during
methyl addition. Previous reports of radiomethyl group addition from
SAM to very long-chain (~48-56 carbons) mycolate precursors by
cell-free supernatants of M. tuberculosis H37Ra suggested
that development of a cell-free system based upon combining recombinant
enzyme produced in Escherichia coli with soluble acceptor fractions from various mycobacterial species would prove successful (9). In addition, we have recently demonstrated that the long meromycolic acid chain is synthesized on an acyl carrier protein designated AcpM by a novel type II fatty acid synthase system (10). To
understand the biosynthetic details of meromycolate modification and
the relationship of this system to the AcpM-utilizing type II fatty
acid synthase system, we developed cell-free systems for studying the
transfer of a methyl group by the meromycolate methyl transferase
enzyme family.
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EXPERIMENTAL PROCEDURES |
Materials and Strains--
M. tuberculosis strain
H37Rv (ATCC 27294), M. smegmatis mc2155
(provided by William R. Jacobs, Albert Einstein College of Medicine, New York) and the various recombinants were grown at 37 °C in Middlebrook 7H9 medium with albumin/dextrose/catalase supplement (ADC)
containing, where appropriated, kanamycin (25 µg/ml) (Sigma) or
hygromycin 50 µg/ml) (Calbiochem). E. coli strain DH5
and JM109 (Life Technologies, Inc.) were used for routine DNA
manipulations and grown in LB broth with, when appropriate, kanamycin
or hygromycin (200 µg/ml) or ampicillin (Sigma) (50 µg/ml). The
electroporation-competent cells of E. coli strain K38 (ATCC
35049) transformed with T7 DNA polymerase-containing plasmid pGP1-2
(11) were prepared by growing at 30 °C in LB with kanamycin to
A600 nm 0.4-0.6. After 1 h in ice, the
500-ml culture was centrifuged and washed twice by prechilled distilled
water and once in 10% cold glycerol. Cells were resuspended in 1.5 ml
of 10% glycerol, and its aliquots were frozen in dry ice-ethanol and
kept at
80 °C.
In addition to the antisera cross-reacting with CMAS-1, MMAS-2, MMAS-3,
and MMAS-4 (7), several monospecific affinity-purified antibodies were
produced from rabbit serum against the following peptides: CMAS-1,
QLVANSENLRSKRV; CMAS-2, YSSNAGWKVERYHRI; MMAS-1, RNHYERSKDRLAAC;
MMAS-4, CAEKPISPTKTRTRFED; AcpM, CPDAVANVQARLEAESK (Quality Controlled
Biochemicals, Hopkinton, MA).
Vectors and Constructs--
PCR primers mma4.1
(5'-gcgcgccatATGACGAGAATGGCCGAGAAACCG) and mma4.2
(5'-ccgcgggctcttccgcaGGCCGCGGCACCCGGCTTGAGG) were used to amplify the
mma4 open reading frame (primer sequence shared by
mma4 is shown in uppercase). These primers introduce an
NdeI site at the start of the open reading frame and a
SapI site after the last codon. The 929-base pair PCR
product was cloned in pCR-Blunt (Invitrogen Corp., San Diego, CA). The
NdeI-SapI fragment was then removed from this
plasmid and inserted into the same sites of pTYB1 (New England Biolabs,
Inc., Beverly, MA) to create the expression plasmid pBGS25. The
E. coli strain ER2566 transformed with pBGS25 was grown at
37 °C in LB containing 100 µg/ml ampicillin to an
OD650 nm of 1.2, then induced with 0.3 mM
isopropyl-
-D-thiogalactopyranoside for 3 h at
30 °C. The predicted molecular mass of the MMAS-4-intein-chitin binding domain fusion protein produced by pBGS25 is 92 kDa. The open
reading frames of genes coding for the methyltransferases CMAS-1,
CMAS-2, MMAS-1, MMAS-2, and MMAS-3 were amplified from the
corresponding cosmids by PCR with Pwo DNA polymerase
(Boehringer Mannheim) using the following pairs of primers:
cma1, 5'-GCGGTACCATGCCCGACGAGCTGAAG-3', 5'-GGAAGCTTGGTGTGCATTGGTAGTCA-3'; cma2,
5'-AGAGAATTCATGCCGATGCAACGGTGG-3', 5'-CGCAGGCTTTTATTTGACCAGAGTG-3';
mma1, 5'-AGAGGTACCATGGCCAAGCTGAGACC-3', 5'-CGCGAATTCGAGCTACTTGGTCATGG-3'; mma2,
5'-AGAGGTACCATGCCTCGAGCATGCG-3', 5'-CGCGAATTCCTACTTCGCCAGCGTG-3';
mma3, 5'-AGAGGTACCATGTCTGATAACTCAACG-3', 5'-GCGGAATTCGTCTACTTGGCCAGCGTG-3'. The PCR products were digested with the proper restriction endonucleases (KpnI and
HindIII for cma1, EcoRI and
HindIII for cma2, KpnI and
EcoRI for mmas) and ligated with pRSET-B
(Invitrogen, Carlsbad, CA) also cut with those enzymes. After
transformation in either DH5
or JM109, plasmids with correct inserts
were selected and sequenced. They were then used to transform strain
K38(pGP1-2) (11) for expressions as His6 tag fusion
proteins using thermal induction. For expression in M. smegmatis, pMH29-cma1, pMH29-cam2,
pMV206-mma1, pMV206-mma2, pMV206-mma3,
and pMV206-mma4 were constructed as described previously (6,
7). Restriction endonucleases, DNA-modifying enzymes, and T4 DNA ligase
were purchased from New England Biolabs. Plasmids or DNA fragments were
purified and isolated with Qiagen plasmid kit, QIAquick nucleotide
removal kit, QIAquick PCR purification kit, or gel extraction kit
(Qiagen Inc., Chatsworth, CA).
Cell-free Assay for Methyltransferase Activity--
Crude cell
lysates were prepared from 1 liter of M. smegmatis cells
grown to an OD650 nm of 0.5-1.0. The cell pellets were
washed with 100 ml of cold Buffer 1 (50 mM potassium
phosphate, pH 7.0, 1 mM dithiothreitol, 1 mM
EDTA) and centrifuged at 12,000 × g at 4 °C for 10 min. The cells were resuspended in 10 ml of Buffer 1 and removed to a
bead beater chamber with 10 g of glass beads (0.1 mm in diameter)
(Biospec Products Inc., Bartlesville, OK). The cells were lysed by
beadbeating twice for 1 min with an intervening 3-min cooling on ice.
The cell lysate was centrifuged twice at 12,000 × g at
4 °C for 10 min and then (where indicated) the lysate was
heat-inactivated at 90 °C for 10 min. In most assays, the
substrate-containing lysate was used on the same day. In some cases
frozen heat-treated substrate was used. Activity from frozen substrate
was comparable with that of fresh lysates over 1-2 weeks. Overproduction of recombinant methyltransferases in K38(pGP1-2) was
induced by growing cells (1:40 dilution from an overnight grown
culture) in 500 ml of LB broth at 30 °C to an
A590 of 0.4. The culture was then incubated with
shaking at 42 °C for 30 min followed by 37 °C for 90 min with
shaking. E. coli cells were washed and lysed as described
for mycobacterial cells. Equal volumes of substrate (mycobacterial
supernatant) and enzyme (E. coli supernatant) were mixed in
a glass vial and incubated with [3H]SAM (American
Radiolabeled Chemicals, Inc., St. Louis, MO) (275 µCi of 72.4 Ci/mmol
per 10-ml reaction) at 37 °C for 1 h. The lipids were
saponified in 10% tetrabutylammonium hydroxide at 100 °C overnight.
Methylation with iodomethane was as described previously (6). The
methyl esters were separated by an acetonitrile to dioxane gradient by
C18 reverse phase HPLC, and radioactivity was monitored by a
-RAM
inline scintillation detector (In/Us System Inc., Fairfield, NJ) (12,
13). Columns were calibrated using methyl esters prepared from
commercially available saturated fatty acids (Aldrich). For the
cell-free assays the following gradient was used: 0-2 min 100%
acetonitrile; 2-52 min linear gradient to 100% dioxane, 52-62 min
hold at 100% dioxane. The extended gradient shown in Fig. 7 was 0-2
min 75/25 acetonitrile/dioxane, 2-62 min linear gradient to 25/75
acetonitrile/dioxane, 62-65 min to 100% dioxane. In inhibition
experiments, the substrates were incubated with 1 mg/ml
S-adenosyl-L-homocysteine (Sigma) or sinefungin
(from Bill Baker, PathoGenesis Corp., Seattle, WA) (17) or rabbit
anti-AcpM polyclonal antisera at 37 °C for 30 min before the
addition of methyltransferase and
[methyl-3H]SAM.
Partial Purification and Identification of the Substrate Complex
for Methyltransferase--
Ammonium sulfate was added to 20%
saturation to the mycobacterial lysate, and the precipitates were
resuspended and dialyzed against distilled water at 4 °C overnight.
The substrate was further purified by Resource Q anion exchange using a
0-1 M NaCl gradient. The sensitivity of the substrates (or
products) to proteinase treatment and alkaline conditions was tested by
incubating the reaction solution with trypsin at 37 °C followed by
the addition of an excess of soybean trypsin inhibitor or by dialysis
against 0.01 N NaOH for 2 h, followed by Folch
extraction (2:1 chloroform:methanol and aqueous partition) to produce
an organic and an aqueous phase. The radioactivity of methylated lipids
of each phase was measured by HPLC. A procedure described previously
(10) was modified and used to detect whether AcpM is the carrier of the
substrate for methyltransferase. The aqueous phase from the cell-free
reaction was lyophilized and then resuspended with 10:10:3
(chloroform:methanol:water). The soluble materials were loaded on a
silica gel TLC plate that was developed with butanol:water:acetic acid
(5:3:2). Pools of materials with different RF value
were scraped from the plate and hydrolyzed with 20% tetrabutylammonium
hydroxide.
Other Procedures--
The Km value of MMAS-2
was measured by the competitive inhibition of the incorporation of
[methyl-3H]SAM with increasing concentrations
of cold SAM. The corrected rates for the reactions were used to
generate a Lineweaver-Burk plot for estimation of the kinetic constants
(14). Optimal reaction pH and the affect of pH on the distribution of
radioactivity in organic and aqueous phases were determined by making
the crude lysate in Buffer 1 with different pH values. For all of these quantitative assays, the peaks corresponding to all long-chain products
were integrated and converted to total counts per 1-h reaction.
Immunogold staining of ultrathin sections was performed with anti-CMAS1
sera as described previously (15).
Meromycolic Acid Preparation--
M. tuberculosis
strain H37Rv was grown at 37 °C to an OD of 1.0 at 650 nm in 7H9
broth with Tween 80 and ADC. The organisms were harvested by
centrifugation at 10,000 × g for 15 min. The resulting
cell pellet was suspended in an equal volume of 40% tetrabutylammonium
hydroxide and incubated at 100 °C overnight. The suspension was then
cooled to room temperature, and an equal volume of 5% iodomethane in
dichloromethane was added. This suspension was mixed on a rotator for
5 h at room temperature and then allowed to sit until phase
separation was complete. The resulting upper phase was discarded. The
lower organic phase was washed with an equal volume of water, then 0.1 N hydrochloric acid, then water again and transferred to a
30-ml Corex centrifuge tube and dried under nitrogen at 70 °C. The
resulting material was solubilized in 10 ml of toluene and 5 ml of
acetonitrile, and the full-length mycolic acids were precipitated by
the further addition of 10 ml of acetonitrile (16). The 25-ml
toluene/acetonitrile solution was chilled to
20 °C for 2 h
and then centrifuged at 12,000 × g for 1 h. The
supernatant was discarded, and the toluene/acetonitrile precipitation
of the mycolic acids was repeated. The precipitated mycolic acids were
then subjected to class separation by preparative silica gel TLC with a
mobile phase of hexane:ethyl acetate (95:5). 1H NMR and
mass spectral analysis of the isolated classes of mycolic acids were in
accord with that reported previously (7).
The meroaldehyde of the various isolated classes was prepared largely
as described previously (17). Briefly, purified methylmethoxy mycolic
acid was placed in a reaction vessel under a vacuum of 26 inches of
mercury and heated to 300 °C for 30 min. The resulting meroaldehyde
was HPLC-purified on a Beckman ODS reversed phase column using an
acetonitrile to dioxane gradient. 1H NMR (300 MHz,
CDCl3):
0.33 (m, 1H), 0.57(m, 1H), 0.64(m, 2H), 0.84(d,
3H), 0.88(t, 3H), 1.25 (br s, >20H), 1.60(br s, 2H), 2.40(t, 2H),
2.93(br s, 1H), 3.30(s, 3H), 9.70(s, 1H). FAB-MS (+) (relative abundance, M + 89 (dioxane adduct)): 919 (35), 947 (100), 975 (65),
1003 (30).
The purified meroaldehyde was oxidized by the addition of silver(II)
nitrate in a solution of 10% sodium hydroxide, 2.5% tetrahydrofuran, 2.5% toluene, and 50% ethanol at room temperature for 1 h. The resulting solution was acidified with hydrochloric acid and extracted three times with diethyl ether. The ether extract was dried under nitrogen at 50 °C. The resulting material containing the mero acid
was solubilized in dichloromethane and methyl-esterified as described
previously (5). The methyl meromethoxy mycolate was purified by
reversed phase HPLC. 1H NMR (500 MHz, CDCl3):
0.33 (m, 1H), 0.57 (m, 1H), 0.64 (m, 2H), 0.84 (d, 3H), 0.88 (t,
3H), 1.25 (br s, >20H), 1.60 (br s, 2H), 2.30 (t, 2H), 2.93 (br s,
1H), 3.33 (s, 3H), 3.70 (s, 3H). The ester was then dried under
nitrogen at 70 °C, hydrolyzed in a solution of 7.5% potassium
hydroxide in 25% methanol at room temperature for 1 h. The
solution was then acidified with hydrochloric acid and extracted with
diethylether three times. The ether extract was dried and subjected to
FAB-MS(
) (relative abundance): 816 (15), 844 (60), 872 (100), 900 (45).
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RESULTS |
Overproduction of the Six Meromycolyl Methyltransferases in E. coli
and M. smegmatis--
We had previously implicated six different
enzymes involved in modification reactions of mycolic acids by
heterologous expression in M. smegmatis and M. tuberculosis. CMAS-1 introduces the distal cis-cyclopropane (5), CMAS-2 introduces the proximal
cis-cyclopropane (6), MMAS-1 converts a cis- to a
trans-olefin with the introduction of an allylic methyl
branch (8), and MMAS-2, MMAS-3, and MMAS-4 together produce the
cis-cyclopropyl methoxymycolic acids (7). To assess the
functions of these enzymes in vitro, we cloned each gene
into one of two E. coli expression vectors, which included either a hexahistidine fusion (His6 tag) at the N terminus
or a chitin-binding domain and intein-containing cleavage sequence at
the C terminus (Fig. 1). Robust
expression of each of these genes was evident with the production of a
fusion protein of the appropriate molecular weight as shown. The
identity of the fusion proteins of CMAS-1, MMAS-1, MMAS-2, MMAS-3, and
MMAS-4 was confirmed by Western blotting using polyclonal antisera
directed to affinity-purified peptide-elicited antisera to a CMAS-1
sequence common to several members of this family of enzymes (data not
shown). Expression of cma1 containing an N-terminal
His6 tag in M. smegmatis and analysis of the
mycolic acids produced by this strain revealed the presence of the
distally cyclopropanated, proximally
-methylated trans-olefinic mycolic acid characterized previously (5),
demonstrating that the presence of the fusion peptide did not adversely
affect enzyme activity (data not shown).

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Fig. 1.
Overproduction of mycolate methyltransferases
in E. coli. Coomassie Brilliant Blue-stained 12.5%
polacrylamide gel electrophoresis of protein lysates prepared from
E. coli strain K38(pGP1-2) containing the pRSETB
hexahistidine fusion proteins with CMAS-1 (lane 1,
uninduced; lane 2, induced), CMAS-2 (lane 3),
MMAS-1 (lane 4), MMAS-2 (lane 5), MMAS-3
(lane 6). The latter samples are all shown induced only;
uninduced samples all appeared similar to lane 1. Lane
7 contains MMAS-4 fused to the chitin binding domain and intein
sequence produced in E. coli strain ER2566.
Arrowheads point to proteins of the correct predicted mass
CMAS-1 (37.1 kDa), CMAS-2 (41.7 kDa), MMAS-1 (37.6 kDa), MMAS-2 (40.2 kDa), MMAS-3 (37.9 kDa), and MMAS-4 (92 kDa). M, molecular
mass standards in kilodaltons.
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We also overproduced each of these enzymes in M. smegmatis
using an efficient expression system based on the plasmids pMH29 and
pMV206 (6). In M. smegmatis, overproduction of each enzyme was less robust, and the phenotype of isolated mycolic acids from these
strains was as described previously.
In Vitro Methyltransferase Activity of the Recombinant
Enzymes--
To evaluate various in vitro assay conditions,
we first reproduced the transfer of tritium from
[methyl-3H]SAM to long-chain fatty acids in
lysates of M. tuberculosis, which had been previously
reported by Takayama and co-workers (9). Using similar conditions we
then labeled clarified wild type M. smegmatis lysates and
discovered that, following saponification and methyl ester formation,
no long-chain components contained radiolabel. Instead, we observed by
reversed phase radio-HPLC an intense peak centering at about 22 min
(Fig. 2A). Presumably this
product corresponds to tuberculostearic acid, the product of
methylation of oleic acid (18). This activity could be completely eliminated by heating the M. smegmatis extract to 90 °C
for 10 min (Fig. 2B). Soluble E. coli extracts
containing any of the methyltransferase enzymes showed no specific
labeling of lipid-containing materials (Fig. 2C), but when
combined with heat-treated (Fig. 2D) or non-heat-treated
(Fig. 2E) lysates from M. smegmatis the recombinant proteins catalyzed the incorporation of a tritium label
from SAM into very long-chain lipid components with HPLC retention
times between 35 and 45 min. The temperature and duration of the heat
treatment were critical to maintaining substrate activity; treatment of
the M. smegmatis lysate at 100 °C for 20 min prior to
addition of the recombinant methyltransferase completed eliminated activity (data not shown).

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Fig. 2.
Definition of the cell-free methyltransferase
assay. All traces represent reversed phase radio-HPLC analysis of
methyl esters prepared from 1-h incubations of the indicated lysate
with [methyl-3H]SAM followed by
saponification, extraction of lipids, and methyl ester formation.
A, M. smegmatis strain mc2155 crude
cell lysate (clarified at 10,000 × g). B,
M. smegmatis crude cell lysate heated at 90 °C for
10 min. C, E. coli (pRSETB_mma2)
crude cell lysate also clarified at 10,000 × g.
D, lysates from B and C mixed
together. E, lysates from A and C
mixed together. F, lysates from B and
C preincubated for 30 min with 1 mg/ml
S-adenosyl-L-homocysteine before adding
[methyl-3H]SAM. In this figure and in Fig. 3,
the gradient used shows the following retention times for saturated
fatty acid methyl esters: C20:0 (14.8 min),
C24:0 (22.2 min), C28:0 (28.8 min),
C30:0 (31.6 min).
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Using heat-inactivated acceptor (90 °C for 10 min) from M. smegmatis and recombinant fusion proteins produced in E. coli, we were able to observe cell-free activity from CMAS-1,
CMAS-2, MMAS-2, MMAS-4, and MMAS-3 + MMAS-4 (Fig.
3, A-F). CMAS-1, CMAS-2, and
MMAS-2 produced similar product distributions (Fig. 3,
B-D), whereas MMAS-4 produced mycolate precursors that
eluted substantially earlier by HPLC (Fig. 3E). The elution
characteristics of this product are consistent with the predicted
introduction of a secondary hydroxy group, rendering the meromycolate
more polar and faster eluting. Interestingly, the presence of the large
intein-chitin binding domain C-terminal sequence did not affect the
ability of this protein to perform its catalytic function. MMAS-1 alone did not result in incorporation of label (not shown), consistent with
the lack of an observable phenotype on heterologous expression of this
enzyme in M. smegmatis (8). Heat-inactivated lysates of
M. tuberculosis did show the incorporation of label into
long-chain precursors when incubated with the recombinant MMAS-1 fusion
protein, again consistent with the phenotypic data (data not shown).
MMAS-3 alone also failed to show in vitro activity,
consistent with the hypothesis that this enzyme requires the
hydroxymethyl meromycolyl precursor produced by MMAS-4. Mixing MMAS-3
and MMAS-4 with M. smegmatis acceptor (Fig. 3F)
produced two series of long-chain products, one coeluting with the
MMAS-4 product and the other significantly less polar (presumably the
corresponding methyl ether).

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Fig. 3.
Cell-free activity from various methyl
transfer enzymes. All traces represent reversed phase radio-HPLC
analysis of saponified methyl esters of fatty acids as in Fig. 2. In
each case a lysate from the indicated recombinant E. coli
strain(s) from Fig. 1 was mixed with a heat-inactivated M. smegmatis lysate prepared as in Fig. 2B. A,
control E. coli lysate; B, CMAS-1; C,
CMAS-2; D, MMAS-2; E, MMAS-4; F,
MMAS-4 + MMAS-3.
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Examination of these assays by evaporative light-scattering detection
showed that the amount of lipid material being radiolabeled was
exceedingly small, suggesting relatively limited pools of such
precursors are available in growing cells.
We could also observe cell-free methyl(en)ation of mycolate precursors
in crude lysates of recombinant M. smegmatis strains overproducing various enzymes (data not shown). In general this activity was more robust than that observed by producing the enzyme and
substrate separately. This system was less useful for further study,
however, because 1) chain length identification was difficult because
of the potential for elongation of precursor molecules by the
endogenous fatty acid synthase II system, and 2) characterization of
the substrate was complicated by the presence of both enzyme and
substrate in the same crude mixture.
Inhibition and Characterization of the in Vitro Methyl Transfer
Reaction--
In vitro methyl(ene) transfer could be
entirely inhibited by low micromolar concentrations of
S-adenosyl-L-homocysteine, the demethylated
analog of SAM (Fig. 2F). This level of inhibition was
similar to that seen with other methyltransferases (19) and was also
observed with another SAM analog, sinefungin (20). Treatment of whole
cells of either M. tuberculosis or various recombinant
M. smegmatis strains with up to millimolar concentrations of
these inhibitors did not affect the mycolic acids produced nor did they
inhibit growth.
The enzymatic reaction was found to be linear over approximately the
first 40 min, and the apparent saturable Km for SAM
was measured as 400 µM. The enzymatic reaction was found to be very pH-sensitive with a dramatic drop in activity between pH 7 and pH 8 (Fig. 4A). This drop
in activity was found to be associated with a change in product
distribution following organic extraction of the completed reaction
with chloroform:methanol (2:1). At moderately acidic pH (between 4 and
6) the bulk of the product remained aqueous-associated following
extraction, whereas at basic pH (between pH 7 and 9) the bulk of the
product was associated with the organic phase (Fig. 4,
B/C). The organic phase following this extraction
contains non-wall-associated lipids, some glycolipids, phospholipids,
and free fatty acids. The aqueous phase contains soluble proteinaceous
material as well as hydrophilic glycolipids and macromolecules. For
products formed at either pH the radio-HPLC profile was identical
following saponification and methyl ester formation. The increase in
organic soluble material at basic pH is consistent with hydrolytic
cleavage of a base-labile linkage of the lipid portion of the substrate
to a proteinaceous carrier (e.g. a thioester linkage).

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Fig. 4.
pH optimization and stability and product
distribution of the cell-free methyltransferase assay.
A, pH versus total product formed by MMAS-2
containing lysates from integration of radio-HPLC tracings expressed as
counts/min. Each pH point contained an identical amount of substrate
containing material and an identical amount of enzyme. B,
aqueous associated counts when the product of the reaction run at the
indicated pH was partitioned between chloroform:methanol (2:1) and
water. C, organic phase-associated counts from the same
extraction, note the lower scale.
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Cellular Localization of the Mycolyl Methyltransferase
Activity--
The enzyme produced in M. smegmatis appeared
to be entirely soluble when analyzed by Western blotting of soluble
lysates and solubilized membrane fractions using antipeptide antisera
that reacted with either CMAS-1 or CMAS-2 (data not shown). The
substrate for methyl transfer also appeared to be soluble, although
some activity could be observed with resuspended membrane fractions. Immunogold localization of the methyltransferases with peptide-based antisera to CMAS-1, which cross-reacts with several of the protein family members, revealed a pronounced bias for membrane association when the proteins were present at natural levels in MTB (Table I). This bias disappeared when the
proteins were overproduced in M. smegmatis, suggesting that,
like cyclopropane fatty acid synthase from E. coli, these
enzymes are predominantly cytoplasmic with a transient, peripheral
association with membrane systems (21).
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Table I
Immunogold localization of mycolate methyltransferases
Ultrathin cryosections were stained with gold-conjugated antipeptide
antisera directed to the CMAS-1 protein, which cross-reacts with
several other members of this protein family. Multiple areas of several
independent sections were quantified by scoring gold particles as being
either not obviously associated with cells (background), associated
with the bacterial cytoplasm, or associated with the cell wall of the
bacteria. The results of two independent experiments with M. smegmatis overproducing CMAS-1 are shown. These two experiments
showed much more intense reaction with the antisera (~10-fold more
labeling), confirming overexpression of the protein. Controls with
non-recombinant M. smegmatis showed essentially no labeling
as did sections reacted with an irrelavent antisera.
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Identification of the Lipid Chain Length during Methyl
Transfer--
The cell-free methyl(en)ation reaction allowed us to
address the question of the length of the lipid during this enzymatic transformation. Simple calibration of the HPLC column revealed a linear
relationship between chain length and retention time as has been
described by previous authors (12, 13). Interpolating from the longest
commercially available saturated fatty acid (30 carbons in length), it
was possible to estimate that the lipid labeled by MMAS-2 in the
cell-free system with a retention time of 45 min possessed a minimum of
40-42 carbons. The introduction of a single unsaturation into the
lipid would have the effect of reducing the retention time (and thus
the apparent chain length) by reversed phase HPLC depending upon the
position of the unsaturated group (9). Full-length meromycolate from
M. tuberculosis would be expected to have the equivalent of
two such desaturations as well as possible methyl ethers or ketones
making the precise retention times difficult to predict. Full-length
mycolic acid, with an extra secondary hydroxy group and an
-alkyl
branch, elutes from this column at about 50 min (Fig.
5A).

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Fig. 5.
Synthesis and HPLC analysis of the
meromycolic acid from the methoxymycolate of M. tuberculosis
H37Rv. A, HPLC analysis with evaporative
light-scattering detection of purified methoxymycolate. The same
gradient as used for the cell-free methyltransferase assay was used for
A and B. B, HPLC analysis of the
meromycolic acid derived from the methoxymycolate series shown in
A. C, synthetic scheme for the pyrolytic cleavage
of the methoxymycolate and oxidation to produce the meromycolic acid
shown in B.
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To address the issue of the retention time of the mono- and
di-unsaturated long-chain mycolate precursors, we synthesized meromycolic acid from full-length mycolic acids purified from the
tubercle bacillus. Pyrolytic cleavage (Fig. 5C) of
full-length mycolate has been described previously and was used in the
initial structural characterization of these molecules (3, 22).
Pyrolysis yielded two major products, a short-chain fatty acid
previously identified as saturated hexacosanoic acid and the long-chain
meroaldehyde. The aldehyde from the methoxymycolate showed the expected
spectral characteristics by 1H NMR (see "Experimental
Procedures") and showed a cluster of ions by FAB-MS of the expected
molecular weight. Oxidation of this aldehyde was accomplished using an
aqueous solution of basic silver nitrate also as described previously
(17). The product of this oxidation was converted to a methyl ester,
and both 1H NMR and FAB-MS data confirmed the predicted
structure. When analyzed by reversed phase HPLC using the same gradient
as for the transferase reaction products, this material was shown to elute at approximately 45 min, 5 min earlier than the full-length mycolic acid and coincident with the longest of the peaks observed in
the in vitro assay system (Figs. 3 and 5B).
The varying values of x, y, and z
(Fig. 5C) combine to make an extremely heterogeneous
collection of lipids that are potential substrates for methyl transfer.
In addition, the question of the chain length of these lipids during
desaturation (and the relative order of desaturation) has not been
adequately addressed. This microheterogeneity of lipid substrates was
obvious when comparing different enzymes operating on the same
substrate pool. Fig. 6A shows
the product distribution when CMAS-1 (which introduces a cyclopropane
into the distal position of the meromycolate) was incubated with
acceptor fractions from heat-inactivated M. smegmatis and
[methyl-3H]SAM. The gradient used to analyze
these samples is shallower in the long-chain region than the previous
gradient to emphasize the complexity of the lipid portions of the
substrates in these reactions. CMAS-1 therefore is capable of
introducing a cyclopropane into lipids shorter than 30 carbons in
length and ranging to full-length meromycolate (56-60 carbons). MMAS-2
(which introduces a cyclopropane into the proximal position) showed a
much narrower product profile with longer chain preference (Fig.
6B), presumably reflecting the fact that the proximal
cyclopropanation cannot occur prior to the introduction of the proximal
olefin, which cannot occur until the chain is at a minimum 36 carbons
in length. Importantly, these results strongly support the proposition
that meromycolate modification occurs concurrent with meromycolate
extension. This differential product formation is not as apparent with
non-heat-inactivated extracts presumably because of continued chain
elongation in those systems.

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Fig. 6.
Analysis of the fine structure of the
radioactive products formed by a proximally cyclopropanating and a
distally cyclopropanating enzyme. A, radio-HPLC trace
of an expanded gradient showing the fine structure of products produced
from the reaction of CMAS-1 operating on the crude heat-inactivated
substrate material from M. smegmatis. B, the same gradient
showing the more limited distribution and longer length of the products
formed by the action of MMAS-2 on the same pool of substrate. The
gradient used shows the following retention times for saturated fatty
acid methyl esters: C20:0 (8.0 min), C24:0
(13.8 min), C28:0 (22.8 min), C30:0 (28.1 min).
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AcpM Is the Lipid Carrier during Methyl Transfer--
Free fatty
acids of even moderate length are relatively insoluble in aqueous
systems and fatty acyl-CoA esters of greater than about 20 carbons are
also highly insoluble. Despite this, the substrates for these methyl
transfer reactions appear to be well behaved soluble molecules. To
understand the nature of the carrier molecule that allows these lipids
to remain soluble, we conducted a series of preliminary trials to
ascertain the identity of this substance. As noted above the carrier
was sensitive to heat at 100 °C for 20 min but was resistant to
90 °C for 10 min. We also found that preincubation of the substrate
with trypsin followed by the addition of excess trypsin inhibitor (but
not mock-incubations) destroyed the ability to incorporate
[3H]SAM into such acceptors. In addition, labeling with
[3H]SAM followed by treatment with trypsin followed by
chloroform:methanol (2:1) to water partition resulted in all of the
radiolabel partitioning into the organic phase, whereas control samples
without added trypsin remained in the aqueous phase. The substrate
could be precipitated by 20% ammonium sulfate treatment and retained
activity following resuspension. The substrate could also be partially purified by ResourceQ anion exchange fast protein liquid
chromatography. The extreme base sensitivity noted above was consistent
with a thioester linkage, and the above results strongly suggested that the carrier molecule was proteinaceous in nature.
We recently identified an acyl carrier protein, AcpM, which accumulates
with attached saturated hexacosanoic acid in the presence of isoniazid
and appears to carry lipids ranging in size to full-length meromycolate
in the absence of drug treatment (10). To test whether AcpM was the
proteinaceous carrier during methyl(ene) transfer in these in
vitro reactions, we preincubated lysates of M. tuberculosis with peptide-based antisera directed toward the C
terminus of AcpM and then added [3H]SAM. Mock-incubations
with irrelevant antisera had no effect on the ability of such extracts
to incorporate label into long-chain lipids, but incubation with
anti-AcpM antisera inhibited such reactions (Fig.
7). Finally, when in vitro
reactions were analyzed by silica gel TLC in the butanol:water:acetic
acid system in which we have previously purified acyl-AcpM, the labeled
product was found to comigrate with purified acyl-AcpM with an
RF of about 0.6.

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Fig. 7.
Inhibition of the cell-free methyltransferase
activity by anti-AcpM antisera. Soluble clarified lysates prepared
from M. tuberculosis H37Rv producing only the normal amounts
of endogenous methyltransferases when incubated with
[methyl-3H]SAM in the absence (A)
or presence (B) of affinity-purified antisera directed to
the C terminus of the AcpM polypeptide. Incubations with irrelevant
antisera (anti-KatG, anti-Acr) did not show any inhibition of the
reaction (not shown).
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DISCUSSION |
Our previous identification of a family of methyltransferases
whose heterologous expression resulted in the modification of full-length mycolic acids allowed the implication of the function of
these enzymes but left open many important questions regarding the
details of the biosynthetic pathway to these important cell wall
constituents (3). Elucidating these details is essential if these
reactions are to be exploited as potential chemotherapeutic targets. In
addition, overproduction of active enzymes was essential for structural
studies of these enzymes to evaluate the hypothesis that a limited
number of active site residues determine product outcome and produce
chemical diversity among mycolic acids. As a first step all six of
these enzymes have been overproduced in E. coli as fusion
proteins, and an in vitro assay has been developed allowing
us to demonstrate that these enzymes retain catalytic activity as
fusion proteins.
In vitro methyl transfer from radiomethyl-SAM to an acceptor
molecule isolated from the mycobacterial cytoplasm has allowed us to
identify both the lipid length of the meromycolate chain during
modification as well as the carrier for mero chain synthesis. Fig.
8 shows a working hypothesis which
integrates this information with the information from an analysis of
the mechanism of action of isoniazid (10). In this model primers for
the type II fatty acid synthase system, which produces mycolic acids,
are produced by the type I fatty acid synthase. These primers are
predominantly palmitate (C16:0) and hexacosanoate
(C26:0). These are then transferred, presumably via
coenzyme A esters, to the acyl carrier protein AcpM. The various
acyl-AcpM molecules are elongated using malonyl-CoA by the type II
system components, which include KasA, KasB, InhA, and MabA (23). The
full 56 carbons of the mero acid do not appear to be completely
synthesized before they are modified. Instead, modification parallels
synthesis and occurs while the acids are attached to the ACP. These
studies do not distinguish the point at which desaturation occurs to
the growing chain and whether desaturation occurs in a parallel fashion
or to a defined substrate that is then elongated by the type II system.
In these heat-inactivated extracts the components of the type II system
are not capable of elongation, therefore the length of the product
formed reflects the length of the substrate used. This has the
interesting implication that the various proximally specific and
distally specific enzymes are measuring the mero chain from the
-end
or, alternatively, that they measure from the thioester end but have a
very wide substrate specificity.

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Fig. 8.
Parallel synthesis and modification of the
-meromycolic acid. This scheme shows the AcpM-bound growing
meromycolic acid chain that is being elongated by the KasA/B-containing
type II fatty acid synthase system and illustrates the wider range of
chain lengths available to CMAS-1 than to CMAS-2 for use in the
cyclopropanation reaction.
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The fact that the in vitro reaction can be inhibited
strongly by antibodies specific for AcpM and that the acceptor has the expected physical properties of an acyl-AcpM strongly suggests that the
growing mero acid remains attached to this ACP during modification and
synthesis. Further evidence for this fact comes from an inspection of
potential desaturases in the mycobacterial genome (24) in which there
appears no clear homologs of a FabA-like dehydrase that produces
cis-olefinic fatty acids. Instead the genome reveals several
potential aerobic mixed function desaturases homologous to plant
enzymes (which act on ACP-bound lipids) but not homologous to
vertebrate enzymes (which act on CoA esters).
These results also confirm the work of Takayama and co-workers who
identified families of monounsaturated, diunsaturated, and
cyclopropanated potential meromycolate intermediates ranging from
26-56 carbons in length (9, 12, 13, 25, 26). The nature of the carrier
molecule during the condensing reaction has not been identified (27),
although we have argued that this is likely to be a simple CoA ester
(3). The ability to specifically label the mero acid product of these
methyltransferases will also allow the details of the condensation step
to be explored.
Several reports have appeared of a particulate system capable of
synthesizing intact mycolates from radio-acetate (although importantly
not from malonate) (28-31). These reports have suggested that all of
the enzymes necessary for mycolic acid production are associated with a
macroparticulate system following partial purification through a
gradient of Percoll. It is difficult to assess the relevance of this
system precisely because: 1) incorporation of acetate but not malonate
or malonyl-CoA would be unusual for an fatty acid synthase system, 2)
in our preparations the meromycolic acid modification enzymes do not
fractionate into this particulate fraction,2 3) in our hands
such fractions contain small numbers of intact or nearly intact
organisms that incorporate acetate very efficiently, and (4) it has not
been possible to inhibit such acetate-dependent activity by
cell-impermeable inhibitors such as sinefungin or S-adenosyl-L-homocysteine. The results presented
here support the proposal that mero acid synthesis occurs largely in
the cell cytoplasm, whereas the growing fatty acyl chain is covalently attached to AcpM via a thioester linkage.
This assay has also allowed us to demonstrate, in principle, that
potential inhibitors can be assessed directly against the appropriate
reaction components, because inhibition could be observed by known
inhibitors of similar enzymes such as
S-adenosyl-L-homocysteine and
sinefungin. Reformatting this assay for high-throughput screening of
potential inhibitors will greatly facilitate the identification of
novel chemotherapeutics for use in the treatment of tuberculosis and
related mycobacterial diseases. In addition, rational drug design using
structural information will be possible following structure elucidation
using these recombinant enzyme overproduction systems.
We thank Debbie Crane for technical and P-3
assistance and Fred Hayes for the immunoelectron microscopy procedures.
We thank Rick Slayden, Khisi Mdluli, and Richard Lee for advice,
technical assistance, purified AcpM samples, and careful proofreading
of the manuscript as well as analytical support.