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 enzym e that catalyzes the final step in the biosynthesis of L-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (B rassica oleracea, var. botrytis) to apparent homogeneity with an overa ll yield of 1.1%, The purification procedure consisted of anion exchan ge, hydrophobic interaction, gel filtration, and fast protein liquid c hromatography, The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophores is and showed a pH optimum for activity between pH 8.0 and 8.5, with a n apparent K-m of 3.3 mM for L-galactono-gamma-lactone. Based on parti al peptide sequence information, polymerase chain reaction fragments w ere isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated, The deduced mature GLDase contain ed 509 amino acid residues with a predicted molecular mass of 57,837 D a, Expression of the cDNA in yeast produced a biologically active prot ein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted with L-galacto no-gamma-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of L-ascorbic a cid biosynthesis in vivo.