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A second GDP-L-galactose phosphorylase in arabidopsis en route to vitamin C. Covalent intermediate and substrate requirements for the conserved reaction
Journal article   Open access   Peer reviewed

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

Carole L Linster, Lital N Adler, Kristofor Webb, Kathryn C Christensen, Charles Brenner and Steven G Clarke
The Journal of biological chemistry, Vol.283(27), pp.18483-18492
07/04/2008
DOI: 10.1074/jbc.M802594200
PMCID: PMC2441562
PMID: 18463094
url
https://doi.org/10.1074/jbc.M802594200View
Published (Version of record) Open Access

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 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.
 Arabidopsis Proteins - genetics Phosphoric Monoester Hydrolases - genetics Arabidopsis - enzymology Galactosephosphates - genetics Substrate Specificity - genetics Ascorbic Acid - genetics Actinidia - genetics Arabidopsis - genetics Arabidopsis Proteins - metabolism Amino Acid Motifs - physiology Ascorbic Acid - biosynthesis Nucleotidyltransferases - genetics Guanosine Diphosphate Sugars - metabolism Actinidia - enzymology Guanosine Diphosphate - genetics Guanosine Diphosphate - metabolism Mutation Nucleotidyltransferases - metabolism Guanosine Diphosphate Sugars - genetics Phosphoric Monoester Hydrolases - metabolism Galactosephosphates - metabolism

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