Publications

• An expression analysis of the ascorbate biosynthesis enzyme VTC2.

  Muller-Moule P.

  The vtc2 mutant of Arabidopsis thaliana is vitamin C deficient and is defective in the ascorbate biosynthesis enzyme GDP-L-Galactose phosphorylase/L -Galactose guanylyltransferase. The connection between the VTC2 gene and this enzyme has only recently been established, and little is known about th ... >> More

  The vtc2 mutant of Arabidopsis thaliana is vitamin C deficient and is defective in the ascorbate biosynthesis enzyme GDP-L-Galactose phosphorylase/L -Galactose guanylyltransferase. The connection between the VTC2 gene and this enzyme has only recently been established, and little is known about the molecular characteristics of the VTC2 gene. In this paper, the expression of the VTC2 gene was characterized on both the RNA and the protein level. The VTC2 gene was expressed in all developmental stages, and the mRNA was expressed at a higher level in green tissues than in the root. VTC2 mRNA expression was induced strongly by exposing dark-grown seedlings to light, to levels higher than found in light-grown seedlings. A VTC2:GUS fusion protein was detected only in green tissues. Unexpectedly, a VTC2:YFP fusion protein was found not only in the cytosol, but also in the nucleus, which suggests that GDP-L-Galactose phosphorylase/L: -Galactose guanylyltransferase might be a dual-function protein, which has both enzymatic and regulatory function. << Less

  Plant Mol. Biol. 68:31-41(2008) [PubMed] [EuropePMC]

• A second GDP-L-galactose phosphorylase in arabidopsis en route to vitamin C. Covalent intermediate and substrate requirements for the conserved reaction.

  Linster C.L., Adler L.N., Webb K., Christensen K.C., Brenner C., Clarke S.G.

  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 d ... >> 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

  J. Biol. Chem. 283:18483-18492(2008) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants.

  Linster C.L., Gomez T.A., Christensen K.C., Adler L.N., Young B.D., Brenner C., Clarke S.G.

  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 stra ... >> More

  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-D-glucose 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. << Less

  J. Biol. Chem. 282:18879-18885(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.

  Dowdle J., Ishikawa T., Gatzek S., Rolinski S., Smirnoff N.

  Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L- ... >> More

  Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis. << Less

  Plant J. 52:673-689(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.