Mechanism and inhibition of the FabV enoyl-ACP reductase from Burkholderia mallei.
Mechanism and inhibition of the FabV enoyl-ACP reductase from Burkholderia mallei.
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DOI：
10.1021/bi902001a
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发表时间：
2010-02-16
期刊：
影响因子：
2.9
通讯作者：
Tonge, Peter J.
中科院分区：
文献类型：
--
作者：
Lu, Hao;Tonge, Peter J.
Enoyl-ACP还原酶催化细菌脂肪酸生物合成(Fas-II)途径延伸周期的最后一步。目前已鉴定出四种不同的Enoyl-ACP还原酶，它们是FabI、FabL、FabK和FabV基因的产物。FabV Enoyl-ACP还原酶是这类酶的最新成员，最初是由Cronan和他的同事在霍乱弧菌中发现的[Massengo-Tiasse和Cronan(2008)霍乱弧菌FabV定义了一类新的Enoyl-ACP载体蛋白还原酶J.Biol。化学。283,1308-1316]。本工作对马来伯克霍尔德氏菌(Burkholderia Mallei)的FabV酶(BmFabV)的作用机理进行了详细的动力学分析，结果表明，bmFabV催化的是先与NADH结合、最后NAD+解离的连续的Bi机制。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)残基以共同的Tyr-(Xaa)8-Lys基序组织。用定点突变研究了这些活性中心残基的作用，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还确定了活性部位(K245)上的第二个赖氨酸，它在Enoyl底物的结合中起着重要作用。由于人们对开发bmFabV抑制剂的兴趣，三氯生对该酶的抑制作用进行了详细的分析，表明三氯生对NADH是竞争性抑制剂，对底物2-十二烯酰辅酶A是非竞争性抑制剂(Ki=0.4µM)。结合荧光结合实验表明，三氯生与酶-NAD+产物复合体结合，与反应路径上的其他中间体处于快速可逆的平衡状态。
Enoyl-ACP reductases catalyze the final step in the elongation cycle of the bacterial fatty acid biosynthesis (FAS-II) pathway. Currently 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 coworkers [Massengo-Tiasse and Cronan (2008) Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase, J. Biol. Chem. 283, 1308–1316]. In the present 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. Due to 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 (Ki = 0.4 µM). Combined with fluorescence binding experiments, it is concluded that triclosan binds to the enzyme-NAD+ product complex which is in rapid and reversible equilibrium with other intermediates on the reaction pathway.
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影响因子：
4.8
作者：
Perozzo, R;Kuo, M;Sacchettini, JC
通讯作者：
Sacchettini, JC
DOI：
10.1006/bbrc.2001.6061
发表时间：
2001-12-14
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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