title: Possible role of a histidine residue in the substrate specificity of yeast d-aspartate oxidase.
abstract: D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids, whereas neutral and basic D-amino acids are substrates of D-amino acid oxidase (DAO). DDO of the yeast Cryptococcus humicola (ChDDO) has much higher substrate specificity to D-aspartate, but the structural features that confer this specificity have not been elucidated. A three-dimensional model of ChDDO suggested that a histidine residue (His56) in the active site might be involved in the unique substrate specificity, possibly through the interaction with the substrate side chain in the active site. His56 mutants with several different amino acid residues (H56A, H56D, H56F, H56K and H56N) exhibited no significant activity toward acidic D-amino acids, but H56A and H56N mutants gained the ability to utilize neutral D-amino acids as substrates, such as D-methionine, D-phenylalanine and D-glutamine, showing the conversion of ChDDO to DAO by these mutations. This conversion was also demonstrated by the sensitivity of these mutants to competitive inhibitors of DAO. These results and kinetic properties of the mutants show that His56 is involved in the substrate specificity of ChDDO and possibly plays a role in the higher substrate specificity toward D-aspartate.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan and shoutaka@vos.nagaokaut.ac.jp.
authors.forename: Shouji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan and.
authors.forename: Kozue
authors.initials: K
authors.lastname: Shimada
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan and.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Nozawa
authors.affiliation: Department of Biomolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Goto
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan and.
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Abe
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan and.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kera
mesh_terms: D021542:Amino Acids, Neutral / Q000737:chemistry; D020134:Catalytic Domain; D003454:Cryptococcus / Q000201:enzymology*; D050804:D-Aspartate Oxidase / Q000737:chemistry* / Q000235:genetics; D026603:D-Aspartic Acid / Q000737:chemistry; D003641:Deamination; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D021542:Amino Acids, Neutral; D005656:Fungal Proteins; D006639:Histidine; D026603:D-Aspartic Acid; D050804:D-Aspartate Oxidase
keywords: Cryptococcus humicola; d-amino acid oxidase; d-aspartate oxidase; flavin oxidase; site-directed mutagenesis; substrate specificity