Mammals, birds, and yeast produce fatty acids via a FAS-I system, a biosynthetic pathway in which each enzymatic activity resides on the single polypeptide chain of a very large multifunctional enzyme (1-3). In contrast, most plants and bacteria utilize a FAS-II system in which each of the enzymatic activities corresponds to individual polypeptides (4 -6). The best characterized FAS-II system is that of Escherichia coli, which includes ␤-ketoacyl-ACP synthases (FabB, FabF, and FabH), a ␤-ketoacyl-ACP reductase (FabG), ␤-hydroxyacyl-ACP dehydrases (FabA and FabZ), and an enoyl-ACP reductase (currently known as FabI and formerly known as EnvM). The E. coli FAS-II system initializes fatty acid biosynthesis by condensing acetyl and malonyl substrates, and then it continues the process by elongating the resulting product through the consecutive addition of C2 units, until the fatty acyl chain reaches a length of about 16 carbons.Some bacteria, such as mycobacteria, possess both a FAS-I and a FAS-II system (7). The mycobacterial FAS-I system displays a bimodal distribution of products centered on C16 and C24 -C26 (7,8). This FAS-II system prefers C16 as a starting substrate (7) and can extend up to C56 (9), indicating that the mycobacterial FAS-II system utilizes the products of the FAS-I system as primers to extend fatty acyl chain lengths even further. The longer chain products of the FAS-II system are the precursors of mycolic acids. Mycolic acids are long chain ␣-alkyl-␤-hydroxy fatty acids, which are a major component of mycobacterial cell walls (10, 11).Isoniazid, a drug used as a first-line antibiotic for the treatment of Mycobacterium tuberculosis infections for the past 40 years, is known to inhibit mycolic acid biosynthesis (12-15). More recently, isoniazid was shown to prevent radiolabel from being incorporated into the chain extension of C24 -C26 fatty acyl substrates (16 -20), suggesting that the target of isoniazid action is within the mycobacterial FAS-II system. Using a genetic approach to isolate the isoniazid target, a single open reading frame was identified (referred to as inhA) in which transformation of the wild-type gene carried on a multicopy plasmid or allelic exchange with a single amino acid substitution mutant (Ser 94 to Ala) was sufficient to confer isoniazid resistance in Mycobacterium smegmatis (21).The protein encoded by the inhA gene, referred to as InhA, has a similar amino acid sequence to two previously characterized enoyl-ACP reductases, namely FabI from E. coli (28% identity) (22)(23)(24)(25), and ENR 1 from Brassica napus (oilseed rape) (23% identity) (26,27). Further analysis revealed that InhA catalyzes the NADH-dependent reduction of the trans double bond between positions C2 and C3 of fatty acyl substrates (28). In addition, InhA prefers fatty acyl substrates of C16 or greater, consistent with its being a member of the mycobacterial FAS-II system (28).The connection between isoniazid action and InhA inhibition is somewhat complicated. Isoniazid is a pro-drug that must be conv...

The crystal structure of the xenobiotic acetyltransferase from Pseudomonas aeruginosa PA103 (PaXAT) has been determined, as well as that of its complex with the substrate chloramphenicol and the cofactor analogue desulfo-coenzyme A. PaXAT is a member of the large hexapeptide acyltransferase family of enzymes that display tandem repeated copies of a six-residue hexapeptide repeat sequence motif encoding a left-handed parallel beta helix (L betaH) structural domain. The xenobiotic acetyltransferase class of hexapeptide acyltransferases is composed of microbial enzymes that utilize acetyl-CoA to acylate a variety of hydroxyl-bearing acceptors. The active site of trimeric PaXAT is a short tunnel into which chloramphenicol and the cofactor analogue desulfo-CoA project from opposite ends. This tunnel is formed by the flat parallel beta sheets of two separate L betaH domains and an extended 39-residue loop. His 79 of the extended loop forms hydrogen bonds from its imidazole NE2 atom to the 3-hydroxyl group of chloramphenicol and from its ND1 group to the peptide oxygen of Thr 86. The interactions of this histidine residue are similar to those found in the structurally unrelated type III chloramphenicol acetyltransferase and suggest that His 79 of PaXAT may be similarly positioned and tautomerically stabilized to serve as a general base catalyst.

A target of the anti-tuberculosis drugs isoniazid (INH) and ethionamide (ETH

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