L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis.
L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.
Department of Vegetable Breeding, National Research Institute of Vegetables, Ornamental Plants and Tea, Mie, Japan. imagic@nivot.affrc.go.jp
T
T
Imai
S
S
Karita
G
G
Shiratori
M
M
Hattori
T
T
Nunome
K
K
Oba
M
M
Hirai
D047151:Amaryllidaceae Alkaloids; D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000235:genetics; D005415:Flavins / Q000032:analysis; D005420:Flavoproteins / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010617:Phenanthridines / Q000494:pharmacology; D018517:Plant Roots / Q000201:enzymology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019657:Solanaceae / Q000201:enzymology / Q000235:genetics*; D013053:Spectrophotometry
D047151:Amaryllidaceae Alkaloids; D018076:DNA, Complementary; D005415:Flavins; D005420:Flavoproteins; D010617:Phenanthridines; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; C015330:lycorine; D001205:Ascorbic Acid