Preparative synthesis of GDP-beta-L-fucose by recombinant enzymes from enterobacterial sources.
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.
Albermann
C
C
Chemische Mikrobiologie, Bergische Universität Wuppertal, Germany.
Distler
J
J
Piepersberg
W
W
D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005643:Fucose / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000302:isolation & purification; D006027:Glycosides / Q000096:biosynthesis*; D006154:Guanosine Diphosphate Fucose / Q000138:chemical synthesis*; D006155:Guanosine Diphosphate Mannose / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D006027:Glycosides; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C110682:wcaG protein, E coli; D006154:Guanosine Diphosphate Fucose; C054686:GDP-4-keto-6-deoxymannose; D005643:Fucose; D006155:Guanosine Diphosphate Mannose; D013401:Sugar Alcohol Dehydrogenases; D007658:Ketone Oxidoreductases; D002238:Carbohydrate Epimerases