Molecular cloning of retinal oxidase/aldehyde oxidase cDNAs from rabbit and mouse livers and functional expression of recombinant mouse retinal oxidase cDNA in Escherichia coli.
Retinal oxidase (EC 1.2.3.11) is a molybdenum-containing flavoenzyme with high enzymatic activity as to retinoic acid synthesis. In this study, we provide direct evidence that retinal oxidase is identical to aldehyde oxidase (EC 1.2.3.1) by cDNA cloning. Retinal oxidase and aldehyde oxidase, purified from rabbit liver cytosol using the original methods, showed completely identical HPLC patterns and amino acid sequences for three corresponding polypeptides (103 amino residues). The primary structural information obtained from the cleaved polypeptides permitted molecular cloning of the full-length cDNA of rabbit liver retinal oxidase (aldehyde oxidase). We also cloned and sequenced the full-length cDNA of mouse retinal oxidase. The cDNAs of rabbit and mouse retinal oxidase have a common sequence approximately 4.6 kb long, comprising 4-kb coding regions. The open reading frames of the cDNAs predict single polypeptides of 1334 and 1333 amino acids; the calculated minimum molecular mass of each is approximately 147,000. Northern blot analysis showed that the rabbit retinal oxidase mRNA was widely expressed in tissues. Finally, we successfully constructed a prokaryotic expression system for mouse retinal oxidase. The purified recombinant retinal oxidase from Escherichia coli showed a typical spectrum of aldehyde oxidases and a lower Km (3.8 microM) for retinal and a higher Vmax (807 nmol/min/mg protein) for retinoic acid synthesis than those of rabbit retinal oxidase (8 microM and 496 nmol/min/mg protein). This represents the first eukaryotic molybdenum-containing flavoprotein to be expressed in an active form in a prokaryotic system.
Huang
D Y
DY
Department of Biochemistry, Faculty of Medicine, Kagawa Medical University, Japan.
Furukawa
A
A
Ichikawa
Y
Y
D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D012160:Retina / Q000201:enzymology*; D050697:Retinal Dehydrogenase; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D003488:Cyanogen Bromide