Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase.
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.
Massengo-Tiassé
R Prisca
RP
Department of Microbiology, University of Illinois, Urbana, Illinois 61801.
Cronan
John E
JE
Department of Microbiology, University of Illinois, Urbana, Illinois 61801; Department of Biochemistry, University of Illinois, Urbana, Illinois 61801. Electronic address: j-cronan@life.uiuc.edu.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D003360:Cosmids; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D005227:Fatty Acids / Q000096:biosynthesis; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000187:drug effects; D014170:Transformation, Genetic / Q000187:drug effects; D014260:Triclosan / Q000494:pharmacology; D014734:Vibrio cholerae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D005227:Fatty Acids; D014260:Triclosan; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)