A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis produces three classes of mycolic acids that differ primarily in the presence and nature of oxygen-containing substituents in the distal portion of the meromycolate branch. The methoxymycolate series has a methoxy group adjacent to a methyl branch, in addition to a cyclopropane in the proximal position. Using the gene for the enzyme that introduces the distal cyclopropane (cma1) as a probe, we have cloned and sequenced a cluster of genes coding for four highly homologous methyl transferases (mma1-4). When introduced into Mycobacterium smegmatis, this gene cluster conferred the ability to synthesize methoxymycolates. By determining the structure of the mycolic acids produced following expression of each of these genes individually and in combination, we have elucidated the biosynthetic steps responsible for the production of the major series of methoxymycolates. The mma4 gene product (MMAS-4) catalyzes an unusual S-adenosyl-L-methionine-dependent transformation of the distal cis-olefin into a secondary alcohol with an adjacent methyl branch. MMAS-3 O-methylates this secondary alcohol to form the corresponding methyl ether, and MMAS-2 introduces a cis-cyclopropane in the proximal position of the methoxy series. The similarity of these reactions and the enzymes that catalyze them suggests that some of the structural diversity of mycolic acids results from different chemical fates of a common cationic intermediate, which in turn results from methyl group addition to an olefinic mycolate precursor.Mycobacterium tuberculosis is directly responsible for more human deaths than any other single infectious agent (1). The intrinsic resistance of this bacterium to many standard chemotherapeutic agents severely limits options for treatment (2). Isoniazid, which targets cell wall biosynthesis, is one of the most effective therapies available, yet it requires prolonged treatment regimens, lowering patient compliance and fostering drug resistance (3). The mycobacterial cell wall is the primary permeability barrier responsible for the intrinsic resistance to many hydrophobic antibiotic substances (4, 5). Understanding the biosynthesis, structure, and function of the mycobacterial cell wall is important, both from the perspective of providing novel drug targets and designing strategies for circumventing intrinsic resistance.The cell wall contains a very high proportion of lipid in the form of mycolic acid, whose physical organization has recently been explored in detail (6, 7). Mycolic acids are long-chain, two-branched, 3-hydroxy fatty acids that range in size in mycobacteria from Ϸ60 to 80 carbons and contain a variety of functional groups on the long (mero) chain (8). These functional groups are either nonpolar moieties, such as olefins, methyl branches, and cyclopropanes, or polar moieties, such as ketones, methoxy groups, epoxides, and esters. The distribution of functional groups among species is distinct and useful for taxonomic identification of species (9, 10). In M. tuberculosis, three...

CD1b-restricted GEM T cell responses are modulated byMycobacterium tuberculosismycolic acid meromycolate chains

“…Strong T cell activation was mediated by the diene mycolic acid MH157, a MA not expressed by Mtb (Table S1) (32). Of the Mtb mycolates, JR1080 induced the strongest T cell activation, which matched the stereochemistry of the expected major Mtb α-mycolate, based on a common biosynthetic pathway for all three major MA classes (33,34). This effect was significantly greater than with the other α-MAs tested, such as MMS131 and MMS130, which are not expressed by Mtb (Table S1).…”

The Mycobacterium tuberculosis cmaA2 Gene Encodes a Mycolic Acid trans-Cyclopropane Synthetase

“…4F and Ref. 8) and a COSY cross-peak between the vinyl protons and a methine proton resonance at 2 ppm (data not shown). cis-Cyclopropanes and cis double bonds in mycolic acids are not adjacent to methyl branches.…”

“…All of these enzymes are known or putative cis-cyclopropane synthetases. PcaA synthesizes the proximal cis-cyclopropane ring of the ␣-mycolates (6), CmaA1 produces a distal cis-cyclopropane ring in the ␣-mycolate of M. smegmatis (15), and MmaA2 likely synthesizes the proximal cis-cyclopropane ring of the methoxymycolates (8,9). Three-dimensional structural studies of these proteins may help elucidate the basis for their substrate specificity.…”

“…The oxygenated mycolic acids contain either cis-or trans-cyclopropane rings at their proximal position. Whereas the putative cis-cyclopropane synthetase of the methoxymycolates has been identified (8,9), the trans-cyclopropane synthetase is unknown. pcaA, one of the members of this distinctive gene family, has been established as essential for M. tuberculosis pathogenesis because a mutant of pcaA cannot establish a chronic persistent M. tuberculosis infection in mice (6).…”

Temperature-dependent Regulation of Mycolic Acid Cyclopropanation in Saprophytic Mycobacteria

“…For instance, the overexpression of M. tuberculosis cmaA1 leads to the conversion of the distal cis double bond into a cis-cyclopropane ring in the ␣-mycolate of M. smegmatis (15). A similar strategy was used to characterize other M. tuberculosis methyltransferases, including CmaA2 (16) and MmaA2 (17). It is noteworthy that the heterologous overexpression studies yielded different results from those obtained in null mutants of the methyltransferase genes in M. tuberculosis, thus leading to conflicting interpretations of the substrate specificities of these enzymes.…”