Characterization of a GDP-D-mannose 3″,5″-epimerase from rice

Article abstract of DOI:10.1016/j.phytochem.2005.12.003

The enzymatic characterization of GDP-d-mannose 3″,5″-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD⁺ in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD⁺ on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K _m and k_cat were determined to be 1.20 × 10 ^-5 M and 0.127 s^-1, respectively, in the presence of 20 μM NAD⁺. The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.