在我们之前的工作中,从氧化葡糖杆菌中鉴定出NAD(H)依赖性羰基还原酶(GoCR),对不同种类的前手性酮的还原表现出中等至高的对映特异性。在本研究中,GoCR的晶体结构是在1.65的分辨率下确定的,并建立了有关底物-酶对接和全原子分子动力学(MD)模拟的计算策略,以帮助理解GoCR的对映参照和对映识别的分子基础,并进一步指导GoCR对映选择性的设计和工程。对于2-氧代-4-苯基丁酸乙酯(OPBE)的还原,三个结合口袋残基,预计Cys93,Tyr149和Trp193在确定对映选择性中起关键作用。通过定点诱变,构建了单点突变体W193A,并证明将OPBE还原为(R)-2-羟基-4-苯基丁酸乙酯(R-HPBE),与野生型(WT)的43.2%相比,ee显着提高>99%。此外,双突变体C93V/Y149A被证明甚至反转GoCR的对映选择性,以79.8%ee提供S-HPBE。
In our previous work, a NAD(H)-dependent carbonyl reductase (GoCR) was identified from Gluconobacter oxydans, which showed moderate to high enantiospecificity for the reduction of different kinds of prochiral ketones. In the present
study, the crystal structure of GoCR was determined at 1.65Å resolution, and a computational strategy concerning substrate-enzyme docking and all-atom molecular dynamics (MD) simulation was established to help understand the molecular basis of enantiopreference and enantiorecognition for GoCR, and to further guide the design and engineering of GoCR enantioselectivity. For the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), three binding pocket residues, Cys93, Tyr149, and Trp193 were predicted to play a critical role in determining the enantioselectivity. Through site-directed mutagenesis, single-point mutant W193A was constructed and proved to reduce OPBE to ethyl (R)-2-hydroxy-4-phenylbutyrate (R-HPBE) with a significantly improved ee of >99% compared to 43.2% for the wild type (WT). Furthermore, double mutant C93V/Y149A was proved to even invert the enantioselectivity of GoCR to afford S-HPBE at 79.8% ee.