Chen Rong, Liu Xu, Wang Jiale, Lin Jinping, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China; Center for Biomedicine and Health, Division of Basical Medicine, Hangzhou Normal University, Hangzhou 310012, China.
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Enzyme Microb Technol. 2015 Mar;70:18-27. doi: 10.1016/j.enzmictec.2014.12.004. Epub 2014 Dec 20.
A new anti-Prelog stereospecific carbonyl reductase (GoKR) from Gluconobacter oxydans DSM2343 was cloned and identified in Escherichia coli. This GoKR formed a homo-tetramer with a subunit size of approximately 27.0kDa. GoKR exhibited full activity with NADPH but not with NADH as a cofactor. The optimal pH and temperature were 9.0 and 30°C, respectively. GoKR reduced various ketones, including aliphatic and aromatic ketones, α- and β-keto esters. Aromatic ketones were reduced to (R)-enantiomers, whereas keto esters were reduced to (S)-hydroxy esters with different enantioselectivities. The data indicate that GoKR does not obey Prelog's rule and exhibits anti-Prelog enantiopreference. Enzyme-substrate-cofactor docking analysis showed that hydride transfer occurred at the si faces of carbonyl group for ethyl 4-chloro-3-oxobutanoate (COBE), which was then selectively reduced to the chiral (S)-alcohol. Excellent enantioselectivities were obtained for reducing COBE and ethyl 2-oxo-4-phenylbutyrate into the corresponding (S)-type products. These products are important for synthesizing HMG-CoA reductase (statins) and angiotensin-converting enzyme inhibitors, respectively.
从氧化葡萄糖酸杆菌DSM2343中克隆并在大肠杆菌中鉴定出一种新的抗普雷洛格立体特异性羰基还原酶（GoKR）。这种GoKR形成了一种同四聚体，亚基大小约为27.0kDa。GoKR以NADPH作为辅因子时表现出完全活性，而以NADH作为辅因子时则无活性。最佳pH值和温度分别为9.0和30°C。GoKR能还原各种酮类，包括脂肪族和芳香族酮、α-和β-酮酯。芳香族酮被还原为（R）-对映体，而酮酯被还原为具有不同对映选择性的（S）-羟基酯。数据表明GoKR不遵循普雷洛格规则，表现出抗普雷洛格对映体偏好。酶-底物-辅因子对接分析表明，对于4-氯-3-氧代丁酸乙酯（COBE），氢化物转移发生在羰基的si面，然后选择性地还原为手性（S）-醇。将COBE和2-氧代-4-苯基丁酸乙酯还原为相应的（S）型产物时获得了优异的对映选择性。这些产物分别对于合成HMG-CoA还原酶（他汀类药物）和血管紧张素转换酶抑制剂很重要。
