title: Implication of a mutation in the flavin binding site on the specific activity and substrate specificity of glycine oxidase from Bacillus subtilis produced by directed evolution.
abstract: Directed evolution was used to expand the substrate specificity and functionality of glycine oxidase by using a high-throughput screening assay based on the 4-aminoantipyrine peroxidase system, with a coefficient of variance below 4%. After screening the library, one mutant with the desired changes was found. The mutant was purified and characterized, showing important changes compared to the wild-type, especially towards cyclic d-amino acids. Amino acid substitution of Ile15 for Val, where the consensus sequence for flavin binding site is placed, seems to be responsible for these changes in specific activity and substrate specificity. The effect of this mutation was explained by using a computer-based three-dimensional model.
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30071 Murcia, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Martínez-Martínez
authors.forename: José
authors.initials: J
authors.lastname: Navarro-Fernández
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Carmona
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sánchez-Ferrer
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003198:Computer Simulation; D019020:Directed Molecular Evolution; D004789:Enzyme Activation; D005415:Flavins / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D005415:Flavins; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase