Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer
Flavocytochrome P450 BM3 mutant W1046A is a NADH-dependent fatty acid hydroxylase: Implications for the mechanism of electron transfer in the P450 BM3 dimer
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DOI：
10.1016/j.abb.2010.09.014
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发表时间：
2011-03-01
影响因子：
3.9
通讯作者：
Munro, Andrew W.
中科院分区：
文献类型：
--
作者：
Girvan, Hazel M.;Dunford, Adrian J.;Munro, Andrew W.
巨大芽孢杆菌P450 BM 3（BM 3）是一种P450/P450还原酶融合酶，其中二聚体被认为是NADPH依赖性脂肪酸羟基化的活性形式。产生BM 3 W1046 A突变体，从其FAD异咯嗪环去除芳香族“屏蔽物”。W1046 A BM 3是一种催化活性的NADH依赖性月桂酸羟化酶，产物形成略上级NADPH驱动的酶。W1046 A BM 3对NADH的Km比野生型BM 3低20倍，并且W1046 A BM 3对NADH和NADPH的催化效率在月桂酸氧化中相似。野生型BM 3也催化NADH依赖性月桂酸羟基化，但效率低于W1046 A BM 3。W1046 A BM 3是无活性的1151的假设有助于支持电子从一个BM 3单体中的FAD转移到另一个BM 3单体中的FMN以驱动天然BM 3中的脂肪酸羟基化的模型。我们的数据显示W1046 A BM 3是一种功能性脂肪酸羟化酶，这与涉及还原酶单体内电子转移以及从一种单体的FMN到另一种单体的血红素的BM 3催化模型一致[12]。W1046 ABM 3是一种高效的脂肪酸羟化酶，具有潜在的生物技术应用价值。(C)2010年爱思唯尔公司All rights reserved.
Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic "shield" from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K-m for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive 1151 helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications. (C) 2010 Elsevier Inc. All rights reserved.