From the Neurobiotechnology Center and the Departments of Biochemistry and Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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Mycobacterium bovis BCG produces a variety of methyl-branched fatty acids. They include C28 to C32 mycocerosic acids esterified to phthiocerol and phenolphthiocerol and the shorter (C22 to C26) mycocerosic acids esterified to phthiocerol. A mycocerosic acid synthase gene-disrupted mutant was still able to produce the shorter mycocerosic acids. The enzyme short chain mycocerosic acid synthase (SMAS), that catalyzes the synthesis of such acids, was purified using anion exchange and red-agarose chromatography. Gel filtration showed the native enzyme to be a 537-kDa protein. Since SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a 280-, 170-, and 100-kDa protein and they cross-reacted with antibodies prepared against the 280- or 100-kDa protein, this enzyme is composed of the three subunits or two 280-kDa monomers with an unusual susceptibility to a proteolytic nick. SMAS utilizes methylmalonyl-CoA with C12 to C20 acyl-CoA as primers and with either NADH or NADPH as the reductant to synthesize the short mycocerosic acids. The Km values for NADH and NADPH were 93 and 90 µM, respectively. Antibodies raised against either the 280- or 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids by SMAS. The enzyme is not immunologically closely related to mycocerosic acid synthase or fatty acid synthase.
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INTRODUCTION |
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Tuberculosis is the world's leading cause of death in humans from a single infectious agent (1). The World Health Organization estimates that Mycobacterium tuberculosis currently afflicts one-third of the world population and will kill 30 million people in this decade (1). In addition, Mycobacterium leprae afflicts 10 to 12 million people worldwide (1). The alarming resurgence of tuberculosis in the United States (2, 3) includes a high percentage of multidrug-resistant tuberculosis which is estimated by WHO sources to already afflict over 50 million people worldwide (1). The bacterial resistance to the frontline antituberculosis drugs, including isoniazid, makes it absolutely critical to find new drugs targeted at other unique processes in the Mycobacteria. The biosynthesis of the unique components of the mycobacterial cell wall might offer such targets.
The Gram-positive hydrophobic mycobacterial cell walls contain up to 60% lipids. The mycobacteria are known to produce unique lipids that make their cell walls an effective barrier to antimicrobial drugs and to the host's natural defense mechanisms and help in their multiplication within the host. Among such unique lipids are the unusually long chain mycolic acids, the very long chain fatty acids that are constituents of the glycopeptidolipids and the methyl-branched fatty acids (4, 5). Common among the methyl-branched acids are tuberculostearic acid (C18 fatty acid) that is found esterified in phosphatidylinositide mannosides, 2,4-dimethyl-C14 acid and mono-, di-, and trimethyl-branched C14-C25 fatty acids found in lipooligosaccharides. Dimethyl-branched fatty acids are found esterified in 2,3-di-O-acyltrehaloses (6). The shorter C22-C26 mycocerosic acids are found esterified to phthiocerol. The tetramethyl-branched C28-C32 mycocerosic acids are found in phenolic glycolipids and esterified to phthiocerol and phenolphthiocerol as well. Finally, the hexa- and heptamethyl-branched phthioceranic and hydroxyphthioceranic acids are also found esterified at various positions to trehalose-2-sulfate to form the sulfolipids that are a characteristic component of the virulent strains of M. tuberculosis.
The enzymology of the biosynthesis of these branched fatty acids remains to be elucidated with one exception. A multifunctional enzyme that catalyzes the synthesis of long chain mycocerosic acids has been purified (7) and the gene that encodes this enzyme has been cloned (8) and disrupted (9). Although the gene-disrupted mutant could not synthesize mycocerosic acids, it produced phthiocerol esters that contained shorter C22 to C26 mycocerosic acids esterified to phthiocerol. Obviously these acids must be produced by an enzyme other than MAS.1 In this paper, we describe the discovery, purification, and characterization of the enzyme which synthesizes the shorter mycocerosic acids from methylmalonyl-CoA and C12 to C20 acyl-CoA primers. We have named this enzyme short chain mycocerosic acid synthase (SMAS).
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Growth Conditions-- TICE BCG vaccine of M. tuberculosis var. bovis BCG was purchased from Organon Teknika-Cappel. The cultures were grown at 37 °C for 8-10 days as described previously (7). mas-mutant (9) was grown in Middlebrook 7H9 medium (Difco) supplemented with ADC (Difco) and 0.05% Tween 80 at 37 °C for 10-12 days in 850-cm plastic roller bottles at 3 rpm in a Roll-in rolling incubator (Bellco Glass).
Isolation of SMAS-- M. tuberculosis BCG cells from 8-10-day-old cultures (30 gm wet cells) were harvested by centrifugation, and suspended in 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM dithioerythritol, 10% glycerol, and 1 mM EDTA (3.0 ml/g of cells) at 4 °C. All subsequent steps in the purification of SMAS were carried out at 4 °C. The cells were disrupted by four passages at 15,000 p.s.i. through a French press (Aminco). The first three passages were immediately preceded by the addition of 1/200 volume of a 0.1 M phenylmethylsulfonyl fluoride (Sigma) solution in isopropyl alcohol. The homogenate was first centrifuged at 37,000 × g for 30 min and the supernatant was then centrifuged at 105,000 × g for 90 min. The final supernatant was used as the cell-free extract.
This solution (600 mg of protein) was loaded onto a DEAE-cellulose (Whatman DE52) column (1.8 × 30 cm) equilibrated with the previously described 0.1 M potassium phosphate buffer containing 1 mM dithioerythritol, pH 7.0. After washing the column with 1 bed volume of the above buffer, a linear gradient of 0.1-0.5 M potassium phosphate buffer, pH 7.0, was applied. Aliquots of the 7.8-ml fractions were assayed for incorporation of [2-14C]methylmalonyl-CoA into lipids using NADH, NADPH, and C20-CoA as the primer. Fractions containing high methylmalonyl-CoA incorporating activity were pooled and concentrated by ultrafiltration using PM-30 membrane (Amicon). This protein solution (4 mg) was dialyzed against 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM dithioerythritol and loaded onto a 6-ml red-agarose (Sigma) column equilibrated with the same phosphate buffer. The unbound protein was removed by washing the column with 1 bed volume of the same buffer, and the bound proteins were eluted by a linear 0.1-0.5 M KCl gradient in the 0.1 M potassium phosphate buffer containing 1 mM dithioerythritol. Aliquots (150 µl) of the 2-ml fractions collected were assayed for methylmalonyl-CoA incorporating activity. Alternatively, the enzyme solution from the DEAE step was loaded onto an Immunopure-protein A immunoglobulin G (IgG) orientation column (Pierce). The column was prepared following the manufacturer's guidelines (Pierce) using 8 mg of total protein of the antisera raised against either the 280-kDa protein or the 100-kDa protein from purified SMAS; 4 mg of protein was loaded onto the column. Protein was eluted using the eluant supplied by the manufacturer following the manufacturer's protocol.Production of Antisera against the 100-kDa Protein and the 280-kDa Protein-- Following separation by SDS-PAGE, the gel segments containing the 100- and 280-kDa proteins were excised, and each was individually injected subcutaneously into New Zealand White male rabbits with Freund's complete adjuvant as described before (10). Three booster injections were administered at 2-week intervals. Blood was collected by heart puncture 4 days after the final injection. The serum was recovered after clot formation.
Electrophoresis and Immunoblotting-- SDS-PAGE was done with a 3% stacking gel and a 5% running gel as described previously (11). Following SDS-PAGE, the proteins were transblotted onto an Immobilon polyvinylidine difluoride membrane (Millipore Corp., Bedford, MA) in 10 mM CAPS buffer, pH 11, and 10% methanol. Subsequent to treatment with 1:250 diluted antisera, the antibody complex was detected by autoradiography using 125I-protein A as the secondary detection agent (12).
Methylmalonyl-CoA Incorporation Assay-- The reaction mixture in a total volume of 500 µl, contained 0.1 M phosphate buffer, pH 6.8, 1 mM NADH, 1 mM NADPH, 2 mM EDTA, 2 mM dithiothreitol, 100 µM acyl-CoA primer, and 25 µM [methyl-14C]methylmalonyl-CoA (56 Ci/mol). For standard assays 0.2 mg of protein was used. Following a 60-min incubation at 37 °C the reaction was stopped by the addition of 250 µl of 10% NaOH and the mixture was heated in a boiling water bath for 10 min; after acidification, the lipids were extracted with chloroform:methanol (2:1). This lipid material was subjected to thin-layer chromatography, with authentic C18 acid as external standard, on Silica Gel G plates using hexane:diethyl ether:formic acid (35:15:1, v/v) as the solvent. After spraying the plates with a 0.1% ethanolic solution of 2,7-dichlorofluorescein, the positions of the UV visible spots were recorded. The plates were scanned for distribution of radioactivity using a Berthold Tracemaster 20 automatic TLC Linear analyzer. Silica gel from the region corresponding to free fatty acids was scraped from the plate into a counting vial, mixed with ScintiverseTM scintillation fluid (Fisher Scientific) and assayed for radioactivity in a Beckman LS3801 Liquid Scintillation System. The enzyme activity is expressed as total picomoles of methylmalonyl-CoA incorporated into free fatty acids/mg of protein.
Product Identification-- To determine if the product generated from methylmalonyl-CoA was free or esterified, after incubation of a standard reaction mixture it was acidified and chloroform-extractable products were removed and analyzed on a TLC plate as indicated above. To determine if any of the product was protein-bound, the aqueous phase that remained after the solvent extraction was boiled for 30 min with 20% NaOH, acidified with 6 M HCl, and the products were extracted with chloroform as indicated above and assayed for 14C.
To determine the chain length of the products generated from methylmalonyl-CoA incorporation, the chloroform-extractable material was subjected to TLC as described before and the fatty acids recovered were refluxed with 14% BF3 in methanol for 3 h at 57 °C. The methyl esters recovered by extraction with chloroform were purified by TLC on Silica Gel G using hexane:diethyl ether:formic acid (35:15:1) as the solvent. Radio-gas liquid chromatography of the methyl esters was performed with a coiled stainless steel column (0.3 × 300 cm) packed with 5% OV-1 (w/w) on Chrom W-HP 80/100 in a Varian 3300 Gas Chromatograph with a 50-300 °C temperature program at 20 °C/min. The effluent from the column was run through a LabLogic GC-RAM detector using Winflow (IN/US Systems) software. A Statistical Deconvolution Algorithm (LabLogic) was used for statistical image enhancement. Chain lengths were determined by comparing retention times of the synthesized products to those of straight chain fatty acid standards.Molecular Weight Estimation-- Gel filtration was carried out on a Sepharose 6B-CL (Pharmacia) (1.5 × 100 cm) column calibrated with goose fatty acid synthase (13), alcohol dehydrogenase (Sigma), bovine serum albumin (Sigma), carbonic anhydrase (Sigma), and cytochrome c (Sigma). The molecular weight of SMAS was calculated from a rectilinear plot of relative mobility versus molecular weight.
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RESULTS |
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Discovery of a Short Chain Mycocerosic Acid Synthase-- The mas-disrupted mutant incorporated labeled propionic acid into C22-C26 mycocerosic acids that are shorter than the C28-C32 mycocerosic acids (9). To seek the enzyme that catalyzes the synthesis of such short mycocerosic acids, cell-free extracts of wild type and mas-disrupted mutant were subjected to DEAE anion exchange chromatography. When the protein fractions were assayed with [methyl-14C]methylmalonyl-CoA and C20-CoA ester as a primer, two major methylmalonyl-CoA incorporating activities were found in the wild type, whereas only one was found in the mas-disrupted mutant (Fig. 1). Fractions 57-67 in the wild type were found to contain MAS. A SDS-PAGE gel and an immunoblot of that gel using antibodies raised against MAS (7) confirmed that MAS was present in fractions 57-67 in the wild type. There was no MAS in the mas-mutant as expected.
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Purification of SMAS--
The 105,000 × g
supernatant prepared from cells of Mycobacterium bovis BCG
was fractionated on DEAE-cellulose columns. Products synthesized by
fractions 33-41 in a methylmalonyl-CoA incorporation assay with
C20-CoA primer firmly established that SMAS was contained in fractions 33-41 obtained from both wild type and
mas-mutant. The DEAE-cellulose fractionation of the high
speed supernatant resulted in a 11-fold purification with complete
recovery of SMAS enzymatic activity (Table
I). The pooled fractions containing SMAS
were concentrated and subjected to red-agarose chromatography. All of
the methylmalonyl-CoA incorporating activity was retained in the column
and upon the application of a linear 0.1-0.5 M KCl the
SMAS activity was eluted as a single peak at 0.2 M KCl. The red-agarose purification step resulted in a 58-fold purification of
SMAS (Table I). This purified enzyme was found to be stable to freezing
in phosphate buffer containing 10% glycerol, and could be stored with
little loss in activity at 70 °C for at least 3 months. The
purified enzyme preparation when subjected to SDS-PAGE revealed three
Coomassie staining bands at 280, 170, and 100 kDa, respectively (Fig.
3A). To test whether the
multiple bands represented subunits or impurities an immunological
approach was used. Rabbit antibodies were raised to the 100-kDa protein
band (Ab100) and the 280-kDa protein band (Ab280). Both antibodies were
individually tested in an immunoaffinity absorption procedure to purify
the SMAS from DEAE-cellulose purified enzyme preparation from the wild
type cells. Ab280 antibody and Ab100 antibody conjugated to two
separate protein A-immobilized matrices were used. The enzyme activity
was retained by both antibodies. Elution of the enzyme from the
affinity matrices at low pH recovered the methylmalonyl-CoA incorporating activity. However, the eluted protein had low enzyme activity. The loss in enzyme activity resulting from the low pH could
be prevented by neutralization of the eluant immediately after it
emerged from the column. SDS-PAGE analysis of the preparation that
resulted from the elution of this activity gave three bands. Using this
procedure SMAS was found to be purified 60-fold, about the same
purification achieved by red-agarose (Table I).
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Molecular Properties of SMAS-- A native molecular mass of 537 kDa was estimated by gel filtration chromatography through a calibrated Sepharose CL-6B column. SDS-PAGE analysis revealed the presence of three bands at 280, 170, and 100 kDa. Therefore it is possible that the native enzyme is a complex composed of these three subunits. The other alternative is that it is a dimer of two 280-kDa monomers and a proteolytic nick was introduced into the monomer during purification.
Ab280 antibodies cross-reacted with all three bands (Fig. 3B) and inhibited the ability of SMAS to incorporate methylmalonyl-CoA into fatty acids in a dose-dependent manner (Fig. 4). Ab100 antibody cross-reacted with all the three bands. However, repeated washings necessary to completely eliminate any background also resulted in the loss of the 280-kDa band that cross-reacted with Ab100 prior to such thorough washing (Fig. 3C). Ab100 also inhibited the ability of the synthase to incorporate methylmalonyl-CoA into fatty acids in a dose-dependent manner (Fig. 4). Antibodies raised against MAS did not inhibit SMAS (Fig. 4). Furthermore, under stringent conditions, the anti-MAS and anti-FAS antibodies failed to cross-react with SMAS on an immunoblot (data not shown). Neither Ab280 nor Ab100 antibody cross-reacted with MAS-containing fractions in an immunoblot. These results clearly show that the SMAS is not immunologically very similar to MAS or FAS.
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Effect of Reductants and Acyl Carrier Protein-- Either NADH or NADPH was found to be sufficient for SMAS activity. The presence of both NADH and NADPH did not enhance the level of incorporation of methylmalonyl-CoA into fatty acids (Table II). NADH alone was slightly more effective than NADPH (Table II). The effect of cofactor concentration on the incorporation of methylmalonyl-CoA into fatty acids by SMAS showed typical Michaelis-Menten saturation patterns for both NADH and NADPH and from double reciprocal plots (Fig. 5) the apparent Km values of 93 and 90 µM were calculated for NADH and NADPH, respectively. Addition of acyl carrier protein did not significantly change the incorporation of methylmalonyl-CoA into fatty acids by SMAS (Table II).
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Effect of Primer Chain Length-- The primer that we routinely used in the assays for SMAS activity was C20-CoA. This primer generated C23 and C26 mycocerosic acids. The ability of SMAS to utilize primers whose chain length varied from C12-CoA to C20-CoA is shown in Table III. SMAS could utilize CoA esters of n-C12, n-C16, n-C18, and n-C20 fatty acids. C16-CoA ester was better than either C12-CoA or C18-CoA and C20-CoA was better than C16-CoA as primer.
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Nature of the Product-- To determine if SMAS releases free fatty acids, the enzymatic reaction products were acidified and partitioned into aqueous (acyl protein) and organic (free fatty acid) phases. About 60% of the labeled product was found in the organic phase. When the products from the organic phase were subjected to TLC, all of the radioactivity in the products was found in the fraction that had an RF identical to that of authentic C18 free fatty acid. Hydrolysis of the aqueous phase released the other 40% of the lipid products as free fatty acids. Radio-gas liquid chromatography of the methyl esters of the methyl-branched fatty acids generated from C20-CoA as the primer, revealed that this enzyme could utilize methylmalonyl-CoA to generate products of chain lengths up to C26 (Fig. 2). Using C16-CoA as the primer, SMAS was again able to synthesize products of chain lengths up to C26. No longer chain length products (mycocerosic acids) could be synthesized.
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DISCUSSION |
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Fatty acid synthases from bacteria such as Escherichia coli are made up of distinct enzymes each of which catalyzes an individual step in the series of reactions involved in fatty acid synthesis (14, 15). Other organisms use multifuctional proteins to catalyze these reactions. Two such multifunctional fatty acid synthesizing enzymes have been found in the mycobacteria. One, fatty acid synthase, utilizes malonyl-CoA to catalyze the synthesis of n-fatty acids (16, 17). The other, mycocerosic acid synthase, utilizes methylmalonyl-CoA to elongate n-fatty acids to generate the multimethyl-branched C28-C32 mycocerosic acids (7). Disruption of the gene that encodes MAS, and the biochemical analysis of the products generated by that mutant, revealed that although the mutant was unable to produce long mycocerosic acids, it was still able to produce other shorter chain C22 to C26 mycocerosic acids (9). Obviously, there must be other enzyme(s) that utilize methylmalonyl-CoA to generate such shorter mycocerosic acids in M. bovis BCG. The discovery, purification, and characterization of such a multifunctional fatty acid synthase described in this paper reveals that although this enzyme shares some properties, as described in the next paragraph, with the other two mycobacterial fatty acid synthesizing enzymes already studied (MAS and FAS), it has its own unique biochemical features.
SMAS appears to be a dimer made up of two 280-kDa monomers. Antibodies prepared against either the 280-kDa protein or the 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids in a dose-dependent manner. Immunoblots using either antibody preparation show the same pattern of three cross-reacting bands at 280, 170, and 100 kDa. It seems likely that a nick in the 280-kDa protein gave rise to the 170- and 100-kDa bands. However, boiling of freshly harvested cells with SDS buffer yielded the same three immunologically cross-reacting bands. These results suggest either that the monomer has unusual susceptibility to proteolytic nick, or, that this multifunctional fatty acid synthesizing enzyme has a unique subunit composition of a 280-, 170-, and a 100-kDa monomer. If so, one set of catalytic domains is present in the 280-kDa protein and another set is distributed between the 170- and 100-kDa proteins, thus with a total of two sets per mole of native enzyme. If the enzyme is composed of two 280-kDa protein monomers, each monomer probably has all of the domains needed for fatty acid synthesis. Thus the overall structure would be like that of a multifunctional dimer as in MAS and FAS.
We have already demonstrated that SMAS can utilize either of the two reductants NADH or NADPH for activity. MAS on the other hand can only utilize NADPH (7). This suggests that the reductase domain is distinct from that found in MAS. While the FAS from mycobacteria releases the products as CoA esters and the product appears to be enzyme-bound in the case of MAS, 60% of the SMAS products are released as free fatty acids; it presumably contains a thioesterase domain. While FAS has no thioesterase domain (20), the acyltransferase domain probably transfers the product to CoA esters. MAS has no thioesterase and the product is enzyme bound. Under standard conditions, SMAS is capable of elongating all four of the acyl-CoA primers tested. FAS is not able to utilize C12 or C14. It is possible that the chain length and structure of the products generated in vivo could depend on the availability of the primers. SMAS could therefore be responsible for the synthesis of the shorter mycocerosic acids found esterified to phthiocerol. SMAS could also be responsible for the synthesis of the multimethyl-branched fatty acids, such as the dimethyl-branched fatty acids found esterified in diacyl trehalose (6), and the mycosanoic acids (18, 19) found in the mycobacteria since they can all be synthesized by elongation of n-fatty acids with methylmalonyl-CoA.
Structural and biochemical relationships among the various types of enzymes that produce the variety of fatty acids in the mycobacteria have recently started to be elucidated (20-22). The amino acid composition showed a similarity between the FAS from M. bovis BCG and Mycobacterium smegmatis and MAS from the former. The gene structure on the other hand revealed the divergence between the FAS and MAS from M. bovis BCG. SMAS appears to be immunologically distinct from any of the mycobacterial enzymes studied thus far. Antibodies raised to FAS (17) and MAS (7) failed to cross-react with SMAS. Gene cloning and sequencing are in progress to determine the structure/domain organization of the SMAS. Obviously, the enzyme must catalyze transacylation steps, condensation, ketoreduction, dehydration, and enoyl reduction. It would be interesting to see whether the gene structure/domain organization resembles the traditional structure seen in FAS (20) or MAS (8). From the fact that SMAS releases its products as free fatty acids, it would appear that the structure would have at least one difference from MAS in that it should possess a thioesterase domain. We have recently discovered that the AT and KS domains of MAS are selective for methylmalonyl-CoA (23). SMAS does not use malonyl-CoA as a substrate and incorporation of methylmalonyl-CoA into short chain mycocerosic acids by SMAS is not inhibited by malonyl-CoA. Since SMAS utilizes methylmalonyl-CoA specifically, the AT and KS domains of this enzyme are likely to be especially homologous to the corresponding domains in MAS.
There has been some question as to whether the pathogenic mycobacteria depend on the host as the main source of fatty acids (24). If they do then it is easy to envision the SMAS elongating the range of fatty acids present in the host to generate some of the methyl-branched fatty acids required for the synthesis of complex wall lipids. The discovery thus far of four unique fatty acid synthesizing and elongating enzymes: MAS, FAS, SMAS, and a fourth methylmalonyl-CoA incorporating activity,2 from M. bovis BCG demonstrates that the synthesis of the wide array of complex lipids is based on a strategy that includes multiple fatty acid synthesizing enzymes. Each class of methyl-branched fatty acids is synthesized by an enzyme dedicated for that purpose. Since the different classes of the methyl-branched fatty acids are channeled to different acyl lipids, the synthesis of the acids may be coupled to esterification to the final acyl acceptor. The use of multiple synthases probably provides the biochemical capability to achieve such channeling. Since the present enzyme is only the second methyl-branched fatty acid synthase to be purified, fuller understanding of the strategies involved must await the elucidation of the nature of the synthases that catalyze the production of the other classes of methyl-branched fatty acids in the mycobacteria.
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FOOTNOTES |
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* This work was supported in part by Grant AI35272 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 614-292-5682;
Fax: 614-292-5379.
1 Tha abbreviations used are: MAS, mycocerosic acid synthase; SMAS, short chain mycocerosic acid synthase; FAS, fatty acid synthase; CAPS, cyclohexylaminopropane sulfonic acid; PAGE, polyacrylamide gel electrophoresis; Ab, antibody.
2 N. D. Fernandes, A. K. Azad, and P. E. Kolattukudy, unpublished data.
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REFERENCES |
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