title: Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.
abstract: Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany).|Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany).
authors.forename: Zhoutong
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany).|Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany).
authors.forename: Richard
authors.initials: R
authors.lastname: Lonsdale
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China).
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Kong
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China).
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China). jiahai@sioc.ac.cn.
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany). reetz@mpi-muelheim.mpg.de.|Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany). reetz@mpi-muelheim.mpg.de.
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077222:Limonene; D008958:Models, Molecular; D015394:Molecular Structure; D013237:Stereoisomerism; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D053138:Cyclohexenes; D013729:Terpenes; D000077222:Limonene; D004851:Epoxide Hydrolases
keywords: amino acid alphabet; directed evolution; epoxide hydrolases; saturation mutagenesis; stereoselectivity