title: Exploring the enantioselective mechanism of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by iterative saturation mutagenesis.
abstract: Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an ER of 65 [WT] to an ES of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
authors: Chao
authors: C
authors: Guo
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
authors: Yanpu
authors: Y
authors: Chen
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
authors: Yu
authors: Y
authors: Zheng
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
authors: Wei
authors: W
authors: Zhang
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
authors: Yunwen
authors: Y
authors: Tao
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China.
authors: Juan
authors: J
authors: Feng
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China lixiatang@uestc.edu.cn.
authors: Lixia
authors: L
authors: Tang
mesh_terms: D060054:Agrobacterium / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D010626:Phenylethyl Alcohol / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D006867:Hydrolases; C430848:halohydrin dehalogenase; D010626:Phenylethyl Alcohol