GDP-Mannose 3′,5′-Epimerase Forms GDP-L-gulose, a Putative Intermediate for the de Novo Biosynthesis of Vitamin C in Plants

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.