title: Towards the computational design and engineering of enzyme enantioselectivity: A case study by a carbonyl reductase from Gluconobacter oxydans.
abstract: 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Kangling
authors.initials: K
authors.lastname: Chen
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Y Adam
authors.initials: YA
authors.lastname: Yuan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: jplin@ecust.edu.cn.
authors.forename: Jinping
authors.initials: J
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: dzhwei@ecust.edu.cn.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000074408:Aldo-Keto Reductases; D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D020600:Gluconobacter oxydans / Q000201:enzymology*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010654:Phenylbutyrates / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010654:Phenylbutyrates; C572920:ethyl (R)-2-hydroxy-4-phenylbutyrate; C572921:ethyl 2-oxo-4-phenylbutyrate; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: Carbonyl reductase; Computational design; Enantiorecognition; Improved enantioselectivity; OPBE