A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals

in Journal of Biological Chemistry (2011), 286(24), 21511-23

The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C ... [more ▼]

The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals. [less ▲]

Defective responses to oxidative stress in protein l-isoaspartyl repair-deficient Caenorhabditis elegans

in Mechanisms of Ageing and Development (2009), 130(10), 670-80

We have shown that Caenorhabditis elegans lacking the PCM-1 protein repair l-isoaspartyl methyltransferase are more sensitive to oxidative stress than wild-type nematodes. Exposure to the redox-cycling ... [more ▼]

We have shown that Caenorhabditis elegans lacking the PCM-1 protein repair l-isoaspartyl methyltransferase are more sensitive to oxidative stress than wild-type nematodes. Exposure to the redox-cycling quinone juglone upon exit from dauer diapause results in defective egg-laying (Egl phenotype) in the pcm-1 mutants only. Treatment with paraquat, a redox-cycling dipyridyl, causes a more severe developmental delay at the second larval stage in pcm-1 mutants than in wild-type nematodes. Finally, exposure to homocysteine and homocysteine thiolactone, molecules that can induce oxidative stress via distinct mechanisms, results in a more pronounced delay in development at the first larval stage in pcm-1 mutants than in wild-type animals. Homocysteine treatment also induced the Egl phenotype in mutant but not wild-type nematodes. All of the effects of these agents were reversed upon addition of vitamin C, indicating that the developmental delay and egg-laying defects result from oxidative stress. Furthermore, we have demonstrated that a mutation in the gene encoding the insulin-like receptor DAF-2 suppresses the Egl phenotype in pcm-1 mutants treated with juglone. Our results support a role of PCM-1 in the cellular responses mediated by the DAF-2 insulin-like signaling pathway in C. elegans for optimal protection against oxidative stress. [less ▲]

A second GDP-L-galactose phosphorylase in Arabidopsis en route to vitamin C covalent intermediate and substrate requirements for the conserved reaction

in Journal of Biological Chemistry (2008), 283(27), 18483-92

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 Smirnoff-Wheeler pathway to plant ... [more ▼]

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 Smirnoff-Wheeler 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. [less ▲]