Institute for Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
Biochemistry. 2010 Feb 16;49(6):1281-9. doi: 10.1021/bi902001a.
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.
烯酰-ACP 还原酶催化细菌脂肪酸生物合成（FAS-II）途径的延伸循环的最后一步。目前，已经鉴定出四种不同的烯酰-ACP 还原酶，它们是 fabI、fabL、fabK 和 fabV 基因的产物。FabV 烯酰-ACP 还原酶是该酶类的最新成员，最初是由 Cronan 及其同事在霍乱弧菌中鉴定出来的[Massengo-Tiasse，R.P.和 Cronan，J.E.（2008）霍乱弧菌 FabV 定义了一种新的烯酰-酰基载体蛋白还原酶类。J. Biol.Chem.283，1308-1316]。在这项工作中，对来自鼻疽伯克霍尔德氏菌（bmFabV）的 FabV 酶的机制进行了详细的动力学分析，结果表明 bmFabV 催化一个顺序双结合机制，首先结合 NADH，最后解离 NAD(+)。该酶是短链脱氢酶/还原酶超家族的成员，其中催化酪氨酸（Y235）和赖氨酸（K244）残基按共识 Tyr-（Xaa）（8）-Lys 基序排列。通过定点突变研究了这些活性位点残基的作用，结果表明 Y235 和 K244 都参与了底物还原过程中的酸碱化学。序列比对和定点突变也鉴定出活性位点中的第二个赖氨酸（K245），它在烯酰底物结合中起重要作用。由于对开发 bmFabV 抑制剂的兴趣，对三氯生对酶的抑制作用进行了详细分析，结果表明三氯生是 NADH 的竞争性抑制剂，是 2-十二烯酰-CoA 的非竞争性抑制剂（K(i) = 0.4 μM）。结合荧光结合实验，我们得出结论，三氯生与酶-NAD(+)产物复合物结合，该复合物与反应途径中的其他中间体处于快速和可逆平衡状态。
