Characterization of a GDP-D-mannose 3'',5''-epimerase from rice.
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 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM 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.
Watanabe
Kentaroh
K
Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen cho 1-1, Sakai, Osaka 599-8531, Japan.
Suzuki
Kiyoshi
K
Kitamura
Shinichi
S
D017360:Arabidopsis / Q000378:metabolism*; D001205:Ascorbic Acid / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D017343:Genes, Plant; D006156:Guanosine Diphosphate Sugars / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
C480160:GDP-gulose; D006156:Guanosine Diphosphate Sugars; D011993:Recombinant Fusion Proteins; D009243:NAD; D002238:Carbohydrate Epimerases; C019192:GDPmannose 3,5-epimerase; D001205:Ascorbic Acid; D005690:Galactose