Stereochemical course and steady state mechanism of the reaction catalyzed by the GDP-fucose synthetase from Escherichia coli.
Recently the genes encoding the human and Escherichia coli GDP-mannose dehydratase and GDP-fucose synthetase (GFS) protein have been cloned and it has been shown that these two proteins alone are sufficient to convert GDP mannose to GDP fucose in vitro. GDP-fucose synthetase from E. coli is a novel dual function enzyme in that it catalyzes epimerizations and a reduction reaction at the same active site. This aspect separates fucose biosynthesis from that of other deoxy and dideoxy sugars in which the epimerase and reductase activities are present on separate enzymes encoded by separate genes. By NMR spectroscopy we have shown that GFS catalyzes the stereospecific hydride transfer of the ProS hydrogen from NADPH to carbon 4 of the mannose sugar. This is consistent with the stereospecificity observed for other members of the short chain dehydrogenase reductase family of enzymes of which GFS is a member. Additionally the enzyme is able to catalyze the epimerization reaction in the absence of NADP or NADPH. The kinetic mechanism of GFS as determined by product inhibition and fluorescence binding studies is consistent with a random mechanism. The dissociation constants determined from fluorescence studies indicate that the enzyme displays a 40-fold stronger affinity for the substrate NADPH as compared with the product NADP and utilizes NADPH preferentially as compared with NADH. This study on GFS, a unique member of the short chain dehydrogenase reductase family, coupled with that of its recently published crystal structure should aid in the development of antimicrobial or anti-inflammatory compounds that act by blocking selectin-mediated cell adhesion.
Menon
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S
Wyeth Research, Cambridge, Massachusetts 02140, USA. smenon@genetics.com
Stahl
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M
Kumar
R
R
Xu
G Y
GY
Sullivan
F
F
D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002384:Catalysis; D002854:Chromatography, Paper; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006154:Guanosine Diphosphate Fucose / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C110682:wcaG protein, E coli; D006153:Guanosine Diphosphate; D006154:Guanosine Diphosphate Fucose; D009249:NADP; D007658:Ketone Oxidoreductases; D002238:Carbohydrate Epimerases