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

Abstract

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.The last missing enzyme of the plant vitamin C biosynthesis pathway was recently identified as a GDP-L-galactose phosphorylase encoded by the Arabidopsis VTC2 gene (1, 2). In this 10-step metabolic pathway from D-glucose to L-ascorbate, GDP-L-Gal 4 phosphorylase catalyzes the formation of L-Gal-1-P in the first committed (and highly regulated) step to vitamin C biosynthesis (2-5). Intriguingly, the VTC2 gene appears to be conserved in vertebrates, which are known to synthesize vitamin C via a different metabolic pathway than plants that does not include GDP-L-Gal as an intermediate (6), and in invertebrates such as Caenorhabditis elegans that may lack the ability to synthesize vitamin C (1). The physiological functions of these apparent orthologs are thus unclear. One clue may be that the Arabidopsis VTC2 enzyme can also use GDP-D-glucose as a substrate (1). Although the role of this activity in plants is unclear, such an activity may be important in other species. Whereas GDP-L-Gal is most probably absent from mammalian tissues, the presence of GDP-D-Glc in bovine mammary gland and the existence of a mammalian GDP-D-Glc pyrophosphorylase have been reported (7,8). More recent observations suggest that GDP-D-Glc formation can be catalyzed by the mammalian GDP-D-mannose pyroph...

A Pathway for Repair of NAD(P)H in Plants

“…The principle of metabolite repair is to increase the fidelity of the enzymatic process, similar to the proofreading activities of DNA polymerases and aminoacyl tRNA synthases in replication and translation. In addition to the pathway described here, other examples of metabolite repair include L-2-hydroxy-glutarate dehydrogenase, which functions to remove the toxic product formed by L-malate dehydrogenase when it acts on ␣-ketoglutarate instead of oxaloacetate (43), and GDP-D-Glc phosphorylase, which compensates for the lack of specificity displayed by GDP-D-Man pyrophosphorylase involved in the formation of glycoconjugates in mammals and worms (44). The importance of L-2-hydroxyglutarate dehydrogenase is emphasized upon its deficiency in humans, which leads to the severe neurometabolic disease L-2-hydroxyglutaric aciduria, increasing the susceptibility to develop brain tumors (45).…”

Extremely Conserved ATP- or ADP-dependent Enzymatic System for Nicotinamide Nucleotide Repair

“…Recent work from our and other laboratories indicates the importance of an emerging group of enzymes serving to eliminate abnormal metabolites that result from side activities of enzymes of the intermediary metabolism (1,2). This variety of metabolite repair enzymes, which we propose to call "metabolite-proofreading enzymes," plays a similar role in intermediary metabolism as the proofreading activities of DNA polymerases and aminoacyl-tRNA synthases in replication and translation, namely to increase the fidelity of the respective biosynthetic processes by correcting for the lack of specificity of the biosynthetic enzymes involved.…”

Ethylmalonyl-CoA Decarboxylase, a New Enzyme Involved in Metabolite Proofreading

“…Control siRNA (Dharmacon ON-TARGETplus non-targeting pool from Thermo Scientific (Epsom, UK)) or ECHDC1 siRNA (Dharmacon ONTARGETplus SMARTpool from Thermo Scientific) was added at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) as the transfection reagent. After 48 -72 h, protein or total RNA was extracted from cells as described previously (16). Protein concentration was determined by the Lowry assay after protein precipitation with 10% trichloroacetic acid, and RNA concentrations were determined by measuring the absorbance at 260 nm.…”

“…siRNA Transfection of HEK293T Cells and Quantitative Real-time PCR-HEK293T cells were maintained and transfected with siRNAs as described (16). Control siRNA (Dharmacon ON-TARGETplus non-targeting pool from Thermo Scientific (Epsom, UK)) or ECHDC1 siRNA (Dharmacon ONTARGETplus SMARTpool from Thermo Scientific) was added at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) as the transfection reagent.…”

“…The activity of this widely distributed stereospecific enzyme, which also acts on NADPHX, is complemented by an NAD(P)HX epimerase (15). Furthermore, a GDP-glucose phosphorylase that corrects a side activity of the GDP-mannose-forming enzyme in mammalian cells and Caenorhabditis elegans has been described (16).…”