The enoyl-[acyl-carrier-protein] reductases FabI and FabL from Bacillus subtilis

Enoyl-[acyl-carrier-protein] (ACP) reductase is a key enzyme in type II fat ty-acid synthases that catalyzes the last step in each elongation cycle. Th e FabI component of Bacillus subtilis (bsFabI) was identified in the genomi c data base by homology to the Escherichia coli protein, bsFabI was cloned and purified and exhibited properties similar to those of E. coli FabI, inc luding a marked preference for NADH over NADPH as a cofactor. Overexpressio n of the B. subtilis fabI gene complemented the temperature-sensitive growt h phenotype of an E. coli fabI mutant. Triclosan was a slow-binding inhibit or of bsFabI and formed a stable bsFabI NAD(+.) triclosan ternary complex. Analysis of the B. subtilis genomic data base revealed a second open readin g frame (ygaA) that was predicted to encode a protein with a relatively low overall similarity to FabI, but contained the Tyr-Xaa(6)-Lys enoyl-ACP red uctase catalytic architecture. The purified YgaA protein catalyzed the NADP H-dependent reduction of trans-2-enoyl thioesters of both N-acetylcysteamin e and ACP. YgaA was reversibly inhibited by triclosan, but did not form the stable ternary complex characteristic of the FabI proteins. Expression of YgaA complemented the fabI(ts) defect in E. coli and conferred complete tri closan resistance. Single knockouts of the ygaA or fabI gene in B. subtilis were viable, but double knockouts were not obtained. The fabI knockout was as sensitive as the wild-type strain to triclosan, whereas the ygaA knocko ut was 250-fold more sensitive to the drug. YgaA was renamed FabL to denote the discovery of a new family of proteins that carry out the enoyl-ACP red uctase step in type II fatty-acid synthases.