Abstract
The D-mannose/L-galactose (D-Man/L-Gal) pathway plays the most important role for ascorbate biosynthesis in higher plants. Many research groups have succeeded in identifying almost all the genes encoding the enzymes in the pathway except the one catalyzing GDP-L-Gal to L-Gal-1P. In this research we set out to characterize and identify the enzyme. Crude extracts prepared from germinating pea seedlings or Arabidopsis leaves showed phosphate-dependent GDP-L-Gal breakdown activities followed by production of GDP and L-Gal-1P, indicating that the reaction was catalyzed by GDP-L-Gal phosphorylase. Because the database analysis indicated that VTC2, a gene isolated from ascorbate-deficient Arabidopsis mutants, had a conserved HIT motif for nucleotide-sugar metabolic enzymes, we suspected that it encoded the enzyme. Three vtc2 alleles showed reduced phosphorylase activities and the recombinant VTC2 protein had GDP-L-Gal phosphorylase activity. These results clearly indicate that VTC2 encodes a novel GDP-L-Gal phosphorylase.
