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 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 DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.
Katane
Masumi
M
Laboratory of Biomolecular Science, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Seida
Yousuke
Y
Sekine
Masae
M
Furuchi
Takemitsu
T
Homma
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism; D050804:D-Aspartate Oxidase / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D050804:D-Aspartate Oxidase; D003605:D-Amino-Acid Oxidase