title: Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.
abstract: Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.
authors.affiliation: Department of Synthetic Organic Chemistry, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany.|Fachbereich Chemie, Philipps-, Universität Marburg, Hans-Meerwein-Strasse, 35032, Marburg, Germany.
authors.forename: Zhoutong
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Synthetic Organic Chemistry, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany.|Fachbereich Chemie, Philipps-, Universität Marburg, Hans-Meerwein-Strasse, 35032, Marburg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Lonsdale
authors.affiliation: Department of Synthetic Organic Chemistry, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany.|Fachbereich Chemie, Philipps-, Universität Marburg, Hans-Meerwein-Strasse, 35032, Marburg, Germany.
authors.forename: Guangyue
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Synthetic Organic Chemistry, 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, 35032, Marburg, Germany. reetz@mpi-muelheim.mpg.de.
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
mesh_terms: D019020:Directed Molecular Evolution; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D016296:Mutagenesis; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D004852:Epoxy Compounds; C013690:styrene oxide; D004851:Epoxide Hydrolases
keywords: directed evolution; epoxide hydrolases; reduced amino acid alphabet; saturation mutagenesis; stereoselectivity