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

Abstract

The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the SmirnoffWheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.Vitamin C (L-ascorbic acid) is the most abundant soluble antioxidant in plants, in which it plays crucial roles in protection against oxidative damage and is used as a cofactor for several enzymes. It is synthesized via reactions initially proposed by Wheeler et al. (1) that are distinct from those used in vitamin C biosynthesis in animals. Although the Smirnoff-Wheeler pathway, arising from GDP-D-mannose and involving L-galactose formation, might not be the only route to vitamin C biosynthesis in plants (alternative pathways arising from myo-inositol and methylgalacturonate or involving L-gulose formation have been proposed; for reviews, see Refs. 2 and 3), it is undoubtedly the pathway that has received the strongest biochemical and genetic support in recent years (4 -9).Of the four loci (VTC1-4) that have been found to be mutated in vitamin C-deficient Arabidopsis thaliana plants (10, 11), three are now known to encode enzymes involved in the Smirnoff-Wheeler pathway. 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 ...

A Novel GDP-d-glucose Phosphorylase Involved in Quality Control of the Nucleoside Diphosphate Sugar Pool in Caenorhabditis elegans and Mammals

“…The activity levels measured in these extracts as the P i -dependent GDP formation from GDP-D-Glc (0.5-3 nmol min Ϫ1 mg of protein Ϫ1 ; see Table 2 and Figs. 1 and 3) were of the same magnitude as the GDP-L-Gal phosphorylase activity measured in Arabidopsis whole plant extracts (24). Under the assay conditions described under "Experimental Procedures," GDP formation measured in the desalted protein extracts in the presence of GDP-D-Glc but 4, respectively, with the assumption that the enzyme preparations were pure.…”

“…GDP-D-Man was, as for plant VTC2 (1), a poor substrate for the human and worm phosphorylases. Also, as for plant VTC2 (24), affinities in the low millimolar range were found with the human and worm enzymes for the nucleotide acceptor P i . These results show that the human C15orf58 and worm C10F3.4 proteins act as specific and highly efficient GDP-D-Glc phosphorylases.…”

Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra)

“…The reverse of GMP expression was observed for GGP (vtc2) in the dark. In the presence of D-mannose 1-phosphate, GMP (vtc1) produces GDP-D-mannose (Wolucka and Van Montagu, 2007), while Arabidopsis GGP shows poor reactivity towards hexose 1-phosphate (Linster et al, 2008). It is possible that the products of both enzymes are channeled for different purposes in plants.…”

Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis

“…The catalytic efficiency of AtGulLO5 (K cat = 0.005 s‒1) is significantly lower compared to that of the other enzymes in the plant AsA pathway such as AtVTC2 (27 s‒1) and AtVTC5 (13 s‒1; [66]). However, it is similar to that of recombinant GME from Arabidopsis (K cat = 0.007 s‒1; [9]).…”