Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian D-aspartate oxidase.
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.
Katane
Masumi
M
Laboratory of Biomolecular Science, Department of Pharmaceutical Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
Saitoh
Yasuaki
Y
Maeda
Kazuhiro
K
Hanai
Toshihiko
T
Sekine
Masae
M
Furuchi
Takemitsu
T
Homma
Hiroshi
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D050804:D-Aspartate Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008322:Mammals / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D000596:Amino Acids; D001120:Arginine; D050804:D-Aspartate Oxidase