caa a22 4500 463185226 CHVBK 20180406164850.0 cr unu---uuuuu 170326e20070101xx s 000 0 eng 10.1007/s00726-006-0350-x doi (NATIONALLICENCE)springer-10.1007/s00726-006-0350-x Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis [Elektronische Daten] [M. Katane, T. Furuchi, M. Sekine, H. Homma] 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. Springer-Verlag, 2006 Keywords: D-aspartate oxidase - Flavoprotein - D-aspartate - Mouse - cDNA cloning - Site-directed mutagenesis nationallicence D-Ala : D-alanine nationallicence D-Asn : D-asparagine nationallicence D-Asp : D-aspartate nationallicence BSA : bovine serum albumin nationallicence DAAO : D-amino acid oxidase nationallicence DASPO : D-aspartate oxidase nationallicence E. coli : Escherichia coli nationallicence FAD : flavin adenine dinucleotide nationallicence FMN : flavin mononucleotide nationallicence D-Glu : D-glutamate nationallicence His-mDASPO : N-terminally His-tagged mouse D-aspartate oxidase nationallicence IPTG : isopropyl-β-D-thiogalactopyranoside nationallicence mDASPO : mouse D-aspartate oxidase nationallicence D-Met : D-methionine nationallicence NMDA : N-methyl-D-aspartate nationallicence NMLA : N-methyl-L-aspartate nationallicence D-Pro : D-proline nationallicence SDS-PAGE : SDS-polyacrylamide gel electrophoresis nationallicence Katane M. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut Furuchi T. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut Sekine M. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut Homma H. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut Amino Acids Springer-Verlag 32/1(2007-01-01), 69-78 0939-4451 32:1<69 2007 32 726 https://doi.org/10.1007/s00726-006-0350-x text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00726-006-0350-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Katane M. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut NATIONALLICENCE 700 1- Furuchi T. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut NATIONALLICENCE 700 1- Sekine M. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut NATIONALLICENCE 700 1- Homma H. Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan aut NATIONALLICENCE 773 0- Amino Acids Springer-Verlag 32/1(2007-01-01), 69-78 0939-4451 32:1<69 2007 32 726 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer