title: Natural diversity to guide focused directed evolution.
abstract: Simultaneous multiple site-saturation mutagenesis was performed at four active-site positions of an esterase from Pseudomonas fluorescens to improve its ability to convert 3-phenylbutyric acid esters (3-PBA) in an enantioselective manner. Based on an appropriate codon choice derived from a structural alignment of 1751 sequences of alpha/beta-hydrolase fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivalent positions. Thus, the number of mutants to be screened could be substantially reduced while the number of functionally intact variants was increased. Whereas the wild-type esterase showed only marginal activity and poor enantioselectivity (E(true)=3.2) towards 3-PBA-ethyl ester, a significant number of hits with improved rates (up to 240-fold) and enantioselectivities (up to E(true)=80) were identified in these "smart" libraries.
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald, Germany.
authors.forename: Helge
authors.initials: H
authors.lastname: Jochens
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010654:Phenylbutyrates / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010654:Phenylbutyrates; C036032:3-phenylbutyric acid; D004950:Esterases