title: Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis.
abstract: A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V (max) K (m) (-1) value for fructosyl valine, the V (max) K (m) (-1) value for fructosyl-(epsilon) N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-(epsilon) N-lysine.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology , Naka-cho, Koganei, Tokyo, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Miura
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferri
authors.forename: Wakako
authors.initials: W
authors.lastname: Tsugawa
authors.forename: Seungsu
authors.initials: S
authors.lastname: Kim
authors.forename: Koji
authors.initials: K
authors.lastname: Sode
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods*; D003920:Diabetes Mellitus / Q000378:metabolism; D006442:Glycated Hemoglobin / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology*; D011487:Protein Conformation; D050879:Sarcosine Oxidase / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000594:Amino Acid Oxidoreductases; D006442:Glycated Hemoglobin; D050879:Sarcosine Oxidase; C517652:hemoglobin A1c protein, human; C103012:amadoriase