Isolation of a cDNA coding for L-galactono-gamma-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants. Purification, characterization, cDNA cloning, and expression in yeast.
L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of L-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1%. The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography. The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent Km of 3.3 mM for L-galactono-gamma-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated. The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da. Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted with L-galactono-gamma-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of L-ascorbic acid biosynthesis in vivo.
Ostergaard
J
J
Laboratorium voor Genetica, Department of Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.mamon@gengenp.rug.ac.be
Persiau
G
G
Davey
M W
MW
Bauw
G
G
Van Montagu
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050544:L-Gulonolactone Oxidase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D051381:Rats; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry
D018076:DNA, Complementary; D010940:Plant Proteins; D010088:Oxidoreductases; D013401:Sugar Alcohol Dehydrogenases; D050544:L-Gulonolactone Oxidase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; D001205:Ascorbic Acid