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Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis

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Summary.

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.

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Abbreviations

D-Ala:

D-alanine

D-Asn:

D-asparagine

D-Asp:

D-aspartate

BSA:

bovine serum albumin

DAAO:

D-amino acid oxidase

DASPO:

D-aspartate oxidase

E. coli :

Escherichia coli

FAD:

flavin adenine dinucleotide

FMN:

flavin mononucleotide

D-Glu:

D-glutamate

His–mDASPO:

N-terminally His-tagged mouse D-aspartate oxidase

IPTG:

isopropyl-β-D-thiogalactopyranoside

mDASPO:

mouse D-aspartate oxidase

D-Met:

D-methionine

NMDA:

N-methyl-D-aspartate

NMLA:

N-methyl-L-aspartate

D-Pro:

D-proline

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

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Authors and Affiliations

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

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

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  1. M. Katane
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  2. T. Furuchi
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  3. M. Sekine
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  4. H. Homma
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Katane, M., Furuchi, T., Sekine, M. et al. Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis. Amino Acids 32, 69–78 (2007). https://doi.org/10.1007/s00726-006-0350-x

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