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Hyperactive mutants of mouse d-aspartate oxidase: mutagenesis of the active site residue serine 308

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Summary.

The role of Ser-308 of murine d-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards d-Asp and N-methyl-d-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.

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Abbreviations

DAAO:

d-amino acid oxidase

DASPO:

d-aspartate oxidase

FAD:

flavin adenine dinucleotide

GHS:

glutathione S-transferase, His, and S

GHS-mDASPO:

N-terminally glutathione S-transferase-, His-, and S-tagged mouse d-aspartate oxidase

GST:

glutathione S-transferase

His-mDASPO:

N-terminally His-tagged mouse d-aspartate oxidase

K d, FAD :

dissociation constant value for binding to flavin adenine dinucleotide

mDASPO:

mouse d-aspartate oxidase

NMDA:

N-methyl-d-aspartate

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Authors and Affiliations

  1. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan

    M. Katane, T. Hanai, T. Furuchi, M. Sekine & H. Homma

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  1. M. Katane
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  2. T. Hanai
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  3. T. Furuchi
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  4. M. Sekine
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  5. H. Homma
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Correspondence to H. Homma.

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Authors’ address: Hiroshi Homma, Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

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Katane, M., Hanai, T., Furuchi, T. et al. Hyperactive mutants of mouse d-aspartate oxidase: mutagenesis of the active site residue serine 308. Amino Acids 35, 75–82 (2008). https://doi.org/10.1007/s00726-007-0627-8

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  • DOI: https://doi.org/10.1007/s00726-007-0627-8

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