title: Pichia stipitis OYE 2.6 variants with improved catalytic efficiencies from site-saturation mutagenesis libraries.
abstract: An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes. Several substrates were sterically demanding, and as hoped, creating additional active site volume yielded better conversions for these alkenes. The most impressive improvement was found for 2-butylidenecyclohexanone. The wild-type provided less than 20% conversion after 24h; a triple mutant afforded more than 60% conversion in the same time period. Moreover, even wild-type OYE 2.6 can reduce cyclohexenones with very bulky 4-substituents efficiently.
authors.affiliation: Department of Chemistry, University of Florida, 126 Sisler Hall, Gainesville, FL 32611, USA.
authors.forename: Athéna
authors.initials: A
authors.lastname: Patterson-Orazem
authors.affiliation: Department of Chemistry, University of Florida, 126 Sisler Hall, Gainesville, FL 32611, USA.
authors.forename: Bradford
authors.initials: B
authors.lastname: Sullivan
authors.affiliation: Department of Chemistry, University of Florida, 126 Sisler Hall, Gainesville, FL 32611, USA. Electronic address: jds2@chem.ufl.edu.
authors.forename: Jon D
authors.initials: JD
authors.lastname: Stewart
mesh_terms: D000475:Alkenes / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology*
chemical_list: D000475:Alkenes; D010088:Oxidoreductases
keywords: Alkene reductase; Flavin; Old yellow enzyme; Reduction