Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis.
L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.
Koshizaka
T
T
Institute of Applied Biochemistry, Yagi Memorial Park, Gifu, Japan.
Nishikimi
M
M
Ozawa
T
T
Yagi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis*; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D006168:Guinea Pigs; D006801:Humans; D050544:L-Gulonolactone Oxidase; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification
D004247:DNA; D013401:Sugar Alcohol Dehydrogenases; D050544:L-Gulonolactone Oxidase; D001205:Ascorbic Acid