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

J Biol Chem. 2000 Dec 22;275(51):40128-33. doi: 10.1074/jbc.M005611200.

Abstract

Enoyl-[acyl-carrier-protein] (ACP) reductase is a key enzyme in type II fatty-acid synthases that catalyzes the last step in each elongation cycle. The FabI component of Bacillus subtilis (bsFabI) was identified in the genomic data base by homology to the Escherichia coli protein. bsFabI was cloned and purified and exhibited properties similar to those of E. coli FabI, including a marked preference for NADH over NADPH as a cofactor. Overexpression of the B. subtilis fabI gene complemented the temperature-sensitive growth phenotype of an E. coli fabI mutant. Triclosan was a slow-binding inhibitor of bsFabI and formed a stable bsFabI.NAD(+). triclosan ternary complex. Analysis of the B. subtilis genomic data base revealed a second open reading 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 reductase catalytic architecture. The purified YgaA protein catalyzed the NADPH-dependent reduction of trans-2-enoyl thioesters of both N-acetylcysteamine 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 triclosan 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 knockout 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 reductase step in type II fatty-acid synthases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacillus subtilis / enzymology*
  • Base Sequence
  • DNA Primers
  • Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)
  • Enzyme Inhibitors / pharmacology
  • Molecular Sequence Data
  • Oxidoreductases / antagonists & inhibitors
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Sequence Homology, Amino Acid
  • Triclosan / pharmacology

Substances

  • DNA Primers
  • Enzyme Inhibitors
  • Triclosan
  • Oxidoreductases
  • Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)