Isolation of a cDNA Coding forl-Galactono-γ-Lactone Dehydrogenase, an Enzyme involved in the Biosynthesis of Ascorbic Acid in Plants

Abstract

L-Galactono-␥-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-␥-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-␥-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of L-ascorbic acid biosynthesis in vivo.

Biosynthesis of Ascorbic Acid in Kidney Bean.l-Galactono-γ-Lactone Dehydrogenase Is an Intrinsic Protein Located at the Mitochondrial Inner Membrane1

“…Previous data from other authors have indicated that the protein responsible for l-GLD activity is located at the mitochondrial fraction of plant cells (Ô ba et al, 1994Mutsuda et al, 1995;Østergaard et al, 1997). However, its exact localization has not as yet been investigated.…”

The Arabidopsis dwarf1 Mutant Is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis1

“…Proteins with known functions include l-gulonolactone oxidase of rat (GULOX_RAT; Koshizaka et al, 1988), l-galactono-1,4-lactone dehydrogenase of Brassica oleracea (DEHYD_BRAOL; Ostergaard et al, 1997), alkyldihydroxyacetonephosphate synthase of human (ADAS_HUMAN; de Vet et al, 1997), berberine-bridge-forming enzyme of Eschscholzia californica (BBE_ECALI; Dittrich and Kutchan, 1991), glycolate oxidase subunit of Helicobacter pylori (GLCOX_HPY; Tomb et al, 1997), and d-lactate dehydrogenase of Archaeoglobus fulgidus (DLDEHYD_AF; Klenk et al, 1997). Mushegian and Koonin (1995) initially proposed that the domain illustrated in Figure 5B is conserved among a broad spectrum of oxidoreductases requiring FAD as a prosthetic group.…”

Ascorbate Biosynthesis in Arabidopsis Cell Suspension Culture

“…The reaction was stopped by adding aliquots to an equal volume of 6% metaphosphoric acid/2.5 mm DTT/1 mm EDTA, containing 2.5 mg L Ϫ1 d-isoascorbic acid as an internal standard. After the samples were centrifuged at 20,000g for 2 min, the l-AA content was analyzed by HPCE, essentially as previously described (Davey et al, 1997). For localization of l-AA biosynthetic activities, cell extracts were centrifuged for 15 min at 10,000g.…”

“…The final oxidation of l-GL to l-AA is catalyzed by the relatively well-characterized enzyme l-galactono-1,4-lactone dehydrogenase (GLDH). GLDH has been purified from the mitochondrial fractions of several sources (Mapson and Breslow, 1958;Ô ba et al, 1994Mutsuda et al, 1995;Østergaard et al, 1997;Imai et al, 1998), and has recently been cloned from cauliflower (Østergaard et al, 1997) and from sweet potato (Imai et al, 1998). The substrate l-GL has also been reported to be a natural component of plant 1 extracts (Østergaard et al, 1997;Wheeler et al, 1998).…”