Cloning and expression of a cDNA encoding mouse kidney D-amino acid oxidase.
A cDNA encoding D-amino acid oxidase (DAO;EC1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5'-terminal untranslated region of 68 nt, an open reading frame of 1035 nt that encodes 345 amino acids (aa), and a 3'-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence, ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr228 and His307, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-beta-D-thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.
Tada
M
M
Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan.
Fukui
K
K
Momoi
K
K
Miyake
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis / Q000235:genetics*; D004274:DNA, Recombinant / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D013552:Swine
D004274:DNA, Recombinant; D003605:D-Amino-Acid Oxidase