Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, UK.
Arch Biochem Biophys. 2011 Mar 1;507(1):75-85. doi: 10.1016/j.abb.2010.09.014. Epub 2010 Sep 22.
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 [15] 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.
巨大芽孢杆菌 P450 BM3（BM3）是一种 P450/P450 还原酶融合酶，其中二聚体被认为是 NADPH 依赖性脂肪酸羟化的活性形式。生成了 BM3 W1046A 突变体，从其 FAD 异咯嗪环中去除了一个芳香“屏蔽”。W1046A BM3 是一种催化活性的 NADH 依赖性月桂酸羟化酶，产物形成略优于 NADPH 驱动的酶。W1046A BM3 对 NADH 的 K(m) 比野生型 BM3 低 20 倍，并且 W1046A BM3 与 NADH 和 NADPH 一起在月桂酸氧化中的催化效率相似。野生型 BM3 也催化 NADH 依赖性月桂酸羟化，但效率低于 W1046A BM3。W1046A BM3 无活性的假设[15]有助于支持一种模型，即电子从一个 BM3 单体中的 FAD 转移到另一个单体中的 FMN，以驱动天然 BM3 中的脂肪酸羟化。我们的数据表明 W1046A BM3 是一种功能性脂肪酸羟化酶，这与涉及还原酶单体内部电子转移的 BM3 催化模型一致，并且从一个单体的 FMN 转移到另一个单体的血红素[12]。W1046A BM3 是一种高效利用 NADH 的脂肪酸羟化酶，具有潜在的生物技术应用。
