Arabidopsis VTC2 Encodes a GDP-l-Galactose Phosphorylase, the Last Unknown Enzyme in the Smirnoff-Wheeler Pathway to Ascorbic Acid in Plants

Abstract

The first committed step in the biosynthesis of L-ascorbate from D-glucose in plants requires conversion of GDP-L-galactose to L-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from A. thaliana as a specific GDP-L-galactose/GDP-Dglucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.Vitamin C (L-ascorbic acid) is well known as an important antioxidant and enzyme cofactor in animals (1, 2) and in plants (3). Apparently, all plants are able to produce vitamin C and mutants completely deficient in synthesis have not been described, suggesting an essential role of ascorbate biosynthesis in these organisms (4). Although vertebrate vitamin C synthesis is restricted to one organ (liver in mammals and kidney in fish, amphibians, and reptiles) (5, 6), virtually all cells in plants can form ascorbate (4). In the few vertebrate species, such as humans, which lack ascorbate biosynthesis, loss of the pathway is compensated by dietary intake, particularly from plants.Different pathways of ascorbate synthesis have evolved in animals and plants. In animals, ascorbate is formed from UDP-D-glucuronate in a pathway involving D-glucuronate formation, reduction and lactonization of D-glucuronate to L-gulonolactone and oxidation of the latter to L-ascorbate (reviewed in Ref. 7). Deficiency of the enzyme catalyzing this last step (L-gulonolactone oxidase) is responsible for the loss of ascorbate synthesis in the vitamin C-requiring vertebrates (8). In plants, the ascorbate synthesis pathway has remained elusive until recently and alternative pathways may exist (9). The Smirnoff-Wheeler pathway (10) has garnered strong biochemical and genetic support (11-16) and appears to represent the major route to ascorbate biosynthesis. In this pathway, GDP-D-mannose, formed from D-mannose 1-phosphate, is successively converted to GDP-L-galactose, L-galactose 1-phosphate, L-galactose, L-galactono-1,4-lactone, and finally to L-ascorbate.Screens for ozone-sensitive (17) or ascorbate-deficient (18) mutants in Arabidopsis thaliana led to the identification of four loci (VTC1, VTC2, VTC3, and VTC4) involved in the maintenance of the vitamin C pool. Characterization of the vtc1 (19) and vtc4 (15) mutants, as well as biochemical studies (14), have allowed the identification of two of the enzymes required for L-ascorbic acid synthesis through the Smirnoff-Wheeler pathway. VTC1 and VTC4 encode GDP-mannose pyrophosphorylase (19) and L-Gal-1-P ...

A Second GDP-l-galactose Phosphorylase in Arabidopsis en Route to Vitamin C

“…Third, does the guanylylated intermediate of VTC2 become resolved by simple phosphorolysis (i.e. transfer to phosphate) as we reported (13) or by transfer to a hexose 1-phosphate as reported by others (14)?…”

“…Cloning of A. thaliana VTC2 and VTC5-The A. thaliana VTC2 cDNA was cloned as described in Linster et al (13). A similar approach was taken to clone the A. thaliana VTC5 cDNA.…”

“…GDP-␤-L-Gal, synthesized and purified as described (17), was provided by Prof. Shinichi Kitamura (Osaka Prefecture University). This preparation was further purified by the reverse-phase HPLC method described in Linster et al (13). Fractions containing GDP-L-Gal were lyophilized, resuspended in H 2 O, and stored at Ϫ20°C.…”

“…Overexpression and Purification of Recombinant VTC2, VTC2 Mutants, and VTC5-Recombinant wild-type and mutant VTC2 enzymes as well as wild-type VTC5 protein were overexpressed and purified as described in Linster et al (13) with the exception that the lysis buffer contained 0.5 mM phenylmethylsulfonyl fluoride and 5 g/ml leupeptin. Protein concentration was determined using a Lowry assay after precipitation with trichloroacetic acid.…”

“…VTC1 encodes GDP-mannose pyrophosphorylase (12), whereasVTC4 encodes L-Gal-1-P 3 phosphatase (8). In 2007 the VTC2 gene product was identified as the enzyme that produces L-Gal-1-P from GDP-L-galactose, which completed the characterization of the 10 enzymatic steps leading from D-glucose to L-ascorbic acid (13,14). VTC2 is a member of the D-galactose-1-phosphate uridylyltransferase/ Apa1 nucleoside monophosphate transferase branch of the histidine triad (HIT) protein superfamily (15).…”