cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex.
The isolation and sequencing of the complete cDNA coding for a d-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time. This 2022 bp cDNA, beside the coding portion, comprises a 5' untranslated tract and the whole 3' region including the polyadenylation signal and the poly(A) tail. The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein. The overexpression of recombinant d-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein. Comparisons with the known cDNAs for mammalian d-amino acid oxidase, another peroxisomal enzyme, are also made. The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level.
Simonic
T
T
Istituto di Fisiologia Veterinaria e Biochimica, Università di Milano, Via Celoria 10, 20133 Milano and Centro Interuniversitario per lo Studio delle Macromolecole Informazionali, Milano, Italy.
Duga
S
S
Negri
A
A
Tedeschi
G
G
Malcovati
M
M
Tenchini
M L
ML
Ronchi
S
S
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D050804:D-Aspartate Oxidase; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D007672:Kidney Cortex / Q000201:enzymology*; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D018076:DNA, Complementary; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase; C494102:DDO protein, human