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-beta-L-
fucose synthetase) to
GDP-beta-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-beta-L-
fucose was optimized and the final product was purified. The formation of
GDP-beta-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-beta-L-
fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg
protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM 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-beta-L-
fucose.