tailieunhanh - Báo cáo khoa học: Caenorhabditis elegans has two genes encoding functional D-aspartate oxidases

Four cDNA clones that were annotated in the database as encoding d-amino acid oxidase (DAAO) or d-aspartate oxidase (DASPO) were iso-lated by RT-PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia colias recombinant proteins with an N-terminal His-tag. | ỊFEBS Journal Caenorhabditis elegans has two genes encoding functional D-aspartate oxidases Masumi Katane Yousuke Seida Masae Sekine Takemitsu Furuchi and Hiroshi Homma Laboratory of Biomolecular Science Schoolof PharmaceuticalSciences Kitasato University Tokyo Japan Keywords Caenorhabditis elegans D-amino acid D-amino acid oxidase D-aspartate oxidase flavoprotein Correspondence H. Homma Laboratory of Biomolecular Science School of Pharmaceutical Sciences Kitasato University 5-9-1 Shirokane Minato-ku Tokyo 108-8641 Japan Fax 81 3 5791 6381 Tel 81 3 5791 6229 E-mail hommah@ These authors contributed equally to this work Note Nucleotide sequence data reported are available in the DDBJ EMBL GenBank databases under the accession numbers AB275890 AB275891 AB275892 and AB275893 Received 17 August 2006 revised 31 October accepted 6 November 2006 doi Four cDNA clones that were annotated in the database as encoding D-amino acid oxidase DAAO or D-aspartate oxidase DASPO were isolated by RT-PCR from Caenorhabditis elegans RNA. The proteins Y69Ap C47Ap F18Ep and F20Hp encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N-terminal His-tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity as it can deaminate neutral and basic D-amino acids whereas the recombinants C47Ap and F18Ep have functional DASPO activities as they can deaminate acidic D-amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates D-Arg more efficiently than D-Ala and D-Met and that C47Ap and F18Ep show distinct kinetic properties against D-Asp D-Glu and N-methyl-D-Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding .