title: GDP-mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants.
abstract: Despite its importance for agriculture, bioindustry, and nutrition, the fundamental process of L-ascorbic acid (vitamin C) biosynthesis in plants is not completely elucidated, and little is known about its regulation. The recently identified GDP-Man 3',5'-epimerase catalyzes a reversible epimerization of GDP-D-mannose that precedes the committed step in the biosynthesis of vitamin C, resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Here, we characterize the native and recombinant GDP-Man 3',5'-epimerase of Arabidopsis thaliana. GDP and GDP-D-glucose are potent competitive inhibitors of the enzyme, whereas GDP-L-fucose gives a complex type of inhibition. The epimerase contains a modified version of the NAD binding motif and is inhibited by NAD(P)H and stimulated by NAD(P)+. A feedback inhibition of vitamin C biosynthesis is observed apparently at the level of GDP-Man 3',5'-epimerase. The epimerase catalyzes at least two distinct epimerization reactions and releases, besides the well known GDP-l-galactose, a novel intermediate: GDP-L-gulose. The yield of the epimerization varies and seems to depend on the molecular form of the enzyme. Both recombinant and native enzymes co-purified with a Hsp70 heat-shock protein (Escherichia coli DnaK and A. thaliana Hsc70.3, respectively). We speculate, therefore, that the Hsp70 molecular chaperones might be involved in folding and/or regulation of the epimerase. In summary, the plant epimerase undergoes a complex regulation and could control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for the cell wall/glycoprotein biosynthesis. Exogenous L-gulose and L-gulono-1,4-lactone serve as direct precursors of l-ascorbic acid in plant cells. We propose an L-gulose pathway for the de novo biosynthesis of vitamin C in plants.
authors.affiliation: Department of Molecular Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB), Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Belgium. beata.wolucka@bio.kuleuven.ac.be
authors.forename: Beata A
authors.initials: BA
authors.lastname: Wolucka
authors.forename: Marc
authors.initials: M
authors.lastname: Van Montagu
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000378:metabolism*; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000737:chemistry; D001667:Binding, Competitive; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002244:Carbon / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006156:Guanosine Diphosphate Sugars / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006601:Hexoses / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: C480160:GDP-gulose; D006156:Guanosine Diphosphate Sugars; D018840:HSP70 Heat-Shock Proteins; D006601:Hexoses; D010455:Peptides; D011994:Recombinant Proteins; C057406:gulose; D002244:Carbon; D005982:Glutathione Transferase; D002238:Carbohydrate Epimerases; C019192:GDPmannose 3,5-epimerase; D009571:Nitrilotriacetic Acid; D001205:Ascorbic Acid