H333N] was significantly more resistant to both antibiotics than FabB and had an affinity for TLM an order of magnitude less than the wild-type enzyme, illustrating that the two-histidine active site architecture is critical to protein-antibiotic interaction. These data provide a structural framework for understanding antibiotic sensitivity within this group of enzymes.

A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the ␤-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso-and anteiso-branchedchain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. coli enzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.

The anaerobic pathway for unsaturated fatty acid synthesis was established in the 1960s in Escherichia coli. The double bond is introduced into the growing acyl chain by FabA, an enzyme capable of both the dehydration of ␤-hydroxydecanoyl-acyl carrier protein (ACP) to trans-2-decenoyl-ACP, and the isomerization of trans-2 to cis-3-decenoyl-ACP. However, there are a number of anaerobic bacteria whose genomes do not contain a fabA homolog, although these organisms nonetheless produce unsaturated fatty acids. We cloned and biochemically characterized a new enzyme in type II fatty acid synthesis from Streptococcus pneumoniae that carries out the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, but is not capable of catalyzing the dehydration of ␤-hydroxy intermediates. This tetrameric enzyme, designated FabM, has no similarity to FabA, but rather is a member of the hydratase/isomerase superfamily. Thus, the branch point in the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation of trans-2-decenoyl-ACP, in contrast to E. coli where the branch point takes place after the formation of ␤-hydroxydecanoyl-ACP.

Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of ␤-ketoacyl-acyl carrier protein (ACP) synthases. The Bacillus subtilis fabF (yjaY) gene (fabF b ) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which B. subtilis adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the B. subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of A universal set of genes encodes the components of the type II, or dissociated, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes (5, 36). The individual chemical transformations are carried out by separate proteins that can be purified independently from other pathway enzymes. The chain elongation steps in fatty acid biosynthesis consist of the condensation of acyl groups, which are derived from acyl-acyl carrier protein (acyl-ACP) or acyl coenzyme A (acyl-CoA), with malonyl-ACP by the ␤-ketoacyl-ACP synthases. These condensing enzymes are divided into two groups. The FabH class of condensing enzymes is responsible for the initiation of fatty acid elongation and utilizes acyl-CoA primers. The FabH of Escherichia coli has been extensively studied, and it selectively uses acetyl-CoA to initiate the pathway (16, 24). In contrast, Bacillus subtilis contains two FabH isozymes that differ from the E. coli enzyme in that they are selective for branchedchain acyl-CoAs (4). The FabB-FabF class of condensing enzymes together catalyze the remaining elongation steps in the pathway (5, 36). These enzymes condense malonyl-ACP with acyl-ACP to extend the acyl chain by two carbons. E. coli expresses both types of condensing enzymes and, although they have overlapping substrate specificity, FabB is responsible for a condensation reaction in unsaturated fatty acid synthesis that cannot be performed by FabF (12,38) and FabF plays a role in the thermal regulation of fatty acid composition (9, 13). Although FabB (32), FabF (21), and FabH (7, 35) all share the same overall structure, the FabB-FabF class of enzymes possesses a Cys-His-His catalytic triad at the active site, whereas the FabH enzymes have a Cys-His-Asn configuration.The two classes of condensing enzymes are also distinguished by their sensitivity to antibiotics. Cerulenin is a fungal epoxide that irreversibly inhi...

The long-chain ␣-alkyl-␤-hydroxy fatty acids, termed mycolic acids, which are characteristic components of the mycobacterial cell wall are produced by successive rounds of elongation catalyzed by a multifunctional (type I) fatty acid synthase complex followed by a dissociated (type II) fatty acid synthase. In bacterial type II systems, the first initiation step in elongation is the condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) catalyzed by ␤-ketoacyl-ACP III (FabH). An open reading frame in the Mycobacterium tuberculosis genome (Rv0533c), now termed mtfabH, was 37.3% identical to Escherichia coli ecFabH and contained the Cys-His-Asn catalytic triad signature. However, the purified recombinant mtFabH clearly preferred longchain acyl-CoA substrates rather than acyl-ACP primers and did not utilize acetyl-CoA as a primer in comparison to ecFabH. In addition, purified mtFabH was sensitive to thiolactomycin and resistant to cerulenin in an in vitro assay. However, mtFabH overexpression in Mycobacterium bovis BCG did not confer thiolactomycin resistance, suggesting that mtFabH may not be the primary target of thiolactomycin inhibition in vivo and led to several changes in the lipid composition of the bacilli. The data presented is consistent with a role for mtFabH as the pivotal link between the type I and type II fatty acid elongation systems in M. tuberculosis. This study opens up new avenues for the development of selective and novel anti-mycobacterial agents targeted against mtFabH.