Department of Chemistry, Zhejiang University, Hangzhou, 310027, China.
Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, 311121, China.
Angew Chem Int Ed Engl. 2019 Oct 7;58(41):14499-14503. doi: 10.1002/anie.201907606. Epub 2019 Sep 5.
Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer-Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild-type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α-keto ester, but with poor yield and stereoselectivity. Structure-guided, site-directed mutagenesis drastically improved the catalytic carbonyl-reduction activity (yield up to 99 %) and stereoselectivity (ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α-keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild-type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations.
环己酮单加氧酶 (CHMO) 对天然 Baeyer-Villiger (BV) 反应具有非常高的催化特异性，迄今为止尚未报道过其具有混杂的还原反应。从不动杆菌属 NCIMB 9871 中发现的野生型 CHMO 具有内在的、混杂的还原芳香族 α-酮酯的能力，但产率和立体选择性较差。基于结构的定点突变极大地提高了催化羰基还原活性（产率高达 99%）和立体选择性（ee 高达 99%），从而将这种 CHMO 转化为酮还原酶，可还原一系列不同取代的芳香族 α-酮酯。与野生型酶相比，突变酶的改进的混杂还原活性源于底物的羰基部分与还原黄素腺嘌呤二核苷酸 (FAD) 辅因子上 N5 上的氢原子之间距离的减小，这一点通过对接和分子动力学模拟得到了证实。
