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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Authors: | M Katane T Furuchi M Sekine H Homma |
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Institution: | (1) Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan |
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Abstract: | 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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Keywords: | : D-aspartate oxidase – Flavoprotein – D-aspartate – Mouse – cDNA cloning – Site-directed mutagenesis |
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