title: Mechanism and inhibition of the FabV enoyl-ACP reductase from Burkholderia mallei.
abstract: Enoyl-ACP reductases catalyze the final step in the elongation cycle of the bacterial fatty acid biosynthesis (FAS-II) pathway. At present, four distinct enoyl-ACP reductases have been identified, which are the products of the fabI, fabL, fabK, and fabV genes. The FabV enoyl-ACP reductase is the most recent member of this enzyme class and was originally identified in Vibrio cholerae by Cronan and co-workers [Massengo-Tiasse, R. P., and Cronan, J. E. (2008) Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase. J. Biol. Chem. 283, 1308-1316]. In this work, a detailed kinetic analysis of the mechanism of the FabV enzyme from Burkholderia mallei (bmFabV) has been undertaken, which reveals that bmFabV catalyzes a sequential bi-bi mechanism with NADH binding first and NAD(+) dissociating last. The enzyme is a member of the short chain dehydrogenase/reductase superfamily in which the catalytic tyrosine (Y235) and lysine (K244) residues are organized in the consensus Tyr-(Xaa)(8)-Lys motif. The role of these active site residues has been investigated using site-directed mutagenesis which has shown that both Y235 and K244 are involved in acid-base chemistry during substrate reduction. Sequence alignment and site-directed mutagenesis also identify a second lysine in the active site (K245) that has an important role in binding of the enoyl substrate. Because of interests in developing inhibitors of bmFabV, a detailed analysis of the inhibition of the enzyme by triclosan has been conducted showing that triclosan is a competitive inhibitor with respect to NADH and an uncompetitive inhibitor with respect to the substrate 2-dodecenoyl-CoA (K(i) = 0.4 muM). In combination with fluorescence binding experiments, we conclude that triclosan binds to the enzyme-NAD(+) product complex which is in rapid and reversible equilibrium with other intermediates on the reaction pathway.
authors.affiliation: Institute for Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Lu
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D042726:Burkholderia mallei / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014260:Triclosan / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D014260:Triclosan; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)