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

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Abstract

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.

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Abbreviations

DAO:

d-Amino acid oxidase

DDO:

d-Aspartate oxidase

FAD:

Flavin adenine dinucleotide

IC50 :

50% Inhibitory concentration

IPTG:

Isopropyl-β-d-thiogalactopyranoside

NMDA:

N-Methyl-d-aspartate

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Acknowledgments

The authors would like to thank Prof. Ryuichi Konno (International University of Health and Welfare) for his kind gift of the mouse DAO expression plasmid, and Mr. Yutaka Taniguchi and Ms. Mayumi Tetsuka, Midori Nitta, and Suno Masakawa for their technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research (21590071) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, as well as by a Kitasato University Research Grant for Young Researchers (to M.K.).

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Authors and Affiliations

  1. Laboratory of Biomolecular Science, Department of Pharmaceutical Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan

    Masumi Katane, Yasuaki Saitoh, Kazuhiro Maeda, Toshihiko Hanai, Masae Sekine, Takemitsu Furuchi & Hiroshi Homma

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  1. Masumi Katane
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  2. Yasuaki Saitoh
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  3. Kazuhiro Maeda
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  4. Toshihiko Hanai
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  5. Masae Sekine
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  6. Takemitsu Furuchi
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  7. Hiroshi Homma
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Correspondence to Hiroshi Homma.

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Katane, M., Saitoh, Y., Maeda, K. et al. Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian d-aspartate oxidase. Amino Acids 40, 467–476 (2011). https://doi.org/10.1007/s00726-010-0658-4

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  • DOI: https://doi.org/10.1007/s00726-010-0658-4

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