Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian d-aspartate oxidase

• Published in: Amino acids Vol. 40; no. 2; pp. 467 - 476
• Main Authors: Katane, Masumi, Saitoh, Yasuaki, Maeda, Kazuhiro, Hanai, Toshihiko, Sekine, Masae, Furuchi, Takemitsu, Homma, Hiroshi
• Format: Journal Article
• Language: English
• Published: Vienna Vienna : Springer Vienna 01.02.2011; Springer Vienna; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amino Acids - metabolism; Analytical Chemistry; Animals; Arginine - chemistry; Arginine - metabolism; Binding Sites; Biochemical Engineering; Biochemistry; Biomedical and Life Sciences; Catalytic Domain; d-Amino acid oxidase; d-Aspartate oxidase; D-Aspartate Oxidase - chemistry; D-Aspartate Oxidase - genetics; D-Aspartate Oxidase - metabolism; Flavoprotein; Kinetics; Life Sciences; Mammals - genetics; Mammals - metabolism; Mice; Models, Molecular; Molecular Sequence Data; Neurobiology; Original Article; Proteomics; site-directed mutagenesis; Substrate Specificity; Amino acid oxidase; Flavoprotein; Site-directed mutagenesis; Substrate specificity; Aspartate oxidase
• ISSN: 0939-4451; 1438-2199
• DOI: 10.1007/s00726-010-0658-4

d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.