title: Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis.
abstract: The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT-PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.
authors.affiliation: Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Katane
authors.forename: T
authors.initials: T
authors.lastname: Furuchi
authors.forename: M
authors.initials: M
authors.lastname: Sekine
authors.forename: H
authors.initials: H
authors.lastname: Homma
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003001:Cloning, Molecular; D050804:D-Aspartate Oxidase / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine
chemical_list: D011993:Recombinant Fusion Proteins; D050804:D-Aspartate Oxidase