title: Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution.
abstract: Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.
authors.affiliation: Institut für Molekulare Biotechnologie, Technische Universität Graz, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Mirela
authors.initials: M
authors.lastname: Ivancic
authors.forename: Goran
authors.initials: G
authors.lastname: Valinger
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
mesh_terms: D001665:Binding Sites; D042725:Burkholderia gladioli / Q000201:enzymology*; D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D004950:Esterases / Q000737:chemistry*; D006885:Hydroxybutyrates / Q000737:chemistry; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D013237:Stereoisomerism
chemical_list: D006885:Hydroxybutyrates; D004950:Esterases