title: Homology modelling and site-directed mutagenesis studies of the epoxide hydrolase from Phanerochaete chrysosporium.
abstract: Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.
authors.affiliation: Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, Chengdu, Sichuan, P. R. China.
authors.forename: Lin-Feng
authors.initials: LF
authors.lastname: Zhang
authors.forename: Jin-Ming
authors.initials: JM
authors.lastname: Wu
authors.forename: Hong
authors.initials: H
authors.lastname: Feng
mesh_terms: D018360:Crystallography, X-Ray; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020075:Phanerochaete / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein
chemical_list: D004851:Epoxide Hydrolases