Glycobiology, 2000, Vol. 10, No. 9 875-881
© 2000 Oxford University Press
Preparative synthesis of GDP-ß-L-fucose by recombinant enzymes from enterobacterial sources
Chemische Mikrobiologie, Bergische Universität Wuppertal, Germany
The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-ß-L-fucose synthetase) to GDP-ß-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-ß-L-fucose was optimized and the final product was purified. The formation of GDP-ß-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-ß-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 µM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 µM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-ß-L-fucose.
1 To whom correspondence should be addressed at: Bergische Universität GH Wuppertal, FB9—Chemische Mikrobiologie, Gauss-Strasse 20, D-42097 Wuppertal, Germany
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