Publications

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

  Heath R.J., Su N., Murphy C.K., Rock C.O.

  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 clone ... >> More

  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. << Less

  J. Biol. Chem. 275:40128-40133(2000) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis.

  Choi K.-H., Heath R.J., Rock C.O.

  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- ... >> More

  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 beta-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-branched-chain 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. << Less

  J. Bacteriol. 182:365-370(2000) [PubMed] [EuropePMC]

  This publication is cited by 22 other entries.

• Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase.

  Massengo-Tiasse R.P., Cronan J.E.

  Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant ... >> More

  Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI. << Less

  J. Biol. Chem. 283:1308-1316(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli.

  Bergler H., Wallner P., Ebeling A., Leitinger B., Fuchsbichler S., Aschauer H., Kollenz G., Hoegenauer G., Turnowsky F.

  The EnvM protein was purified from an overproducing Escherichia coli strain. It showed NADH-dependent enoyl-acyl carrier protein (ACP) reductase activity using both crotonyl-ACP and crotonyl-CoA as substrates. The protein bound a radioactive diazaborine derivative in the presence of NAD+ and radio ... >> More

  The EnvM protein was purified from an overproducing Escherichia coli strain. It showed NADH-dependent enoyl-acyl carrier protein (ACP) reductase activity using both crotonyl-ACP and crotonyl-CoA as substrates. The protein bound a radioactive diazaborine derivative in the presence of NAD+ and radioactive NAD+ in the presence of the drug. Based on these data, it is concluded that EnvM is the NADH-dependent enoyl-ACP reductase (EC 1.3.1.9) of E. coli and we propose to rename the corresponding gene fabI. << Less

  J. Biol. Chem. 269:5493-5496(1994) [PubMed] [EuropePMC]

• Enoyl-acyl carrier protein reductase (fabI) plays a determinant role in completing cycles of fatty acid elongation in Escherichia coli.

  Heath R.J., Rock C.O.

  The role of enoyl-acyl carrier protein (ACP) reductase (E.C. 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli. All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl ... >> More

  The role of enoyl-acyl carrier protein (ACP) reductase (E.C. 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli. All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl-CoA to butyryl-ACP in vitro were purified. These proteins were malonyl-CoA:ACP transacylase (fabD), beta-ketoacyl-ACP synthase III (fabH), beta-ketoacyl-ACP reductase (fabG), beta-hydroxydecanoyl-ACP dehydrase (fabA), and enoyl-ACP reductase (fabI). Unlike the other enzymes in the cycle, FabA did not efficiently convert its substrate beta-hydroxybutyryl-ACP to crotonyl-ACP, but rather the equilibrium favored formation of beta-hydroxybutyryl-ACP over crotonyl-ACP by a ratio of 9:1. The amount of butyryl-ACP formed depended on the amount of FabI protein added to the assay. Extracts from fabI(Ts) mutants accumulated beta-hydroxybutyryl-ACP, and the addition of FabI protein to the fabI(Ts) extract restored both butyryl-ACP and long-chain acyl-ACP synthesis. FabI was verified to be the only enoyl-ACP reductase required for the synthesis of fatty acids by demonstrating that purified FabI was required for the elongation of both long-chain saturated and unsaturated fatty acids. These results were corroborated by analysis of the intracellular ACP pool composition in fabI(Ts) mutants that showed beta-hydroxybutyryl-ACP and crotonyl-ACP accumulated at the nonpermissive temperature in the same ratio found in the fabI(Ts) extracts and in the in vitro reconstruction experiments that lacked FabI. We conclude that FabI is the only enoyl-ACP reductase involved in fatty acid synthesis in E. coli and that the activity of this enzyme plays a determinant role in completing cycles of fatty acid biosynthesis. << Less

  J. Biol. Chem. 270:26538-26542(1995) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.