L-Galactono- -lactone Dehydrogenase from Sweet Potato: Purification and cDNA Sequence Analysis

Abstract

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.

Vitamin C biosynthesis in trypanosomes: A role for the glycosome

“…Extrapolation of the slopes allowed kinetic constants for both metabolites to be calculated ( Table 1). The K M value toward L-galactono-␥-lactone is comparable to those of plant GALDHs (60-150 M) (18,28,29). TbALO exhibited a higher affinity and activity toward D-arabinono-␥-lactone than to L-galactono-␥-lactone, as indicated by its higher apparent V max , turnover constant, catalytic efficiency, and lower apparent K M value.…”

“…2). This is similar to plant GALDHs, where leucine is found at this site, leading to the suggestion that plant enzymes may interact noncovalently with FAD (28,29). In the related flavoprotein vanillyl-alcohol oxidase (VAO), the equivalent histidine facilitates flavinylation at a second histidine residue near the C terminus (30).…”

Arabidopsis CSN5B Interacts with VTC1 and Modulates Ascorbic Acid Synthesis

“…AsA is synthesized via multiple biosynthetic pathways, including the D-glucosone (Loewus, 1999), D-galacturonate (Davey et al, 1999), myo-inositol (Lorence et al, 2004), and D-Man/L-Gal pathways (Wheeler et al, 1998); of these, the D-Man/L-Gal pathway is particularly important in plants (Wheeler et al, 1998;Lorence et al, 2004;Ishikawa and Shigeoka, 2008). The enzymes involved in these pathways have been identified (Imai et al, 1998;Conklin et al, 1999;Gatzek et al, 2002;Wolucka and Van Montagu, 2003;Laing et al, 2004Laing et al, , 2007Dowdle et al, 2007). Mutations in the D-Man/L-Gal pathway result in significantly decreased AsA synthesis.…”

An Arabidopsis Purple Acid Phosphatase with Phytase Activity Increases Foliar Ascorbate

“…Entry points for the biosynthesis of AsA that have been characterized include GDP-Man and L-Gal (Wheeler et al, 1998), myoinositol and D-GlcUA (Lorence et al, 2004), D-GalUA (Agius et al, 2003), and L-gulose (Wolucka and Van Montagu, 2003). All genes of the GDP-Man pathway have been characterized, including those encoding GDP-D-Man pyrophosphorylase (Conklin et al, 1999), GDP-Man-3#,5#-epimerase (Wolucka and Van Montagu, 2003), L-Gal dehydrogenase (Gatzek et al, 2002), L-galactono-1,4-lactone dehydrogenase (Imai et al, 1998), L-Gal-1-P phosphatase (Laing et al, 2004), and, most recently, two genes that encode enzymes that convert GDP-L-Gal to L-Gal-1-P (Dowdle et al, 2007;Laing et al, 2007;Linster et al, 2007).…”