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

摘要

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 K-m 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.