Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase
Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase
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DOI：
10.1002/cbic.201600484
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发表时间：
2016-12-14
期刊：
影响因子：
3.2
通讯作者：
Bornscheuer, Uwe T.
中科院分区：
文献类型：
--
作者：
Beier, Andy;Bordewick, Sven;Bornscheuer, Uwe T.
Baeyer-Villiger单加氧酶（BVMOs）利用分子氧和辅助因子催化酮氧化成酯或内酯。I型BVMOs对NADPH表现出强烈的偏好。然而，对于工业用途来说，NADH是首选的辅因子，因为它便宜十倍，而且更稳定。因此，我们从不动杆菌sp. NCIMB 9871 （CHMOAcineto）中创建了环己酮单加氧酶的变体；这比NADPH使用NADH好4200倍。通过结构分析、序列比对和文献资料相结合，鉴定了辅助因子附近的21个残基，并将其作为诱变目标。与NADPH（58%）相比，携带3或4个突变的两个组合变体显示出更高的环己酮与NADH的转化（79%）以及特异性。导致I型BVMO辅因子特异性改变的结构原因是NADH的两个羟基通过直接相互作用和桥接水分子来协调氢键网络。
Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79%) compared to NADPH (58%) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.