1093384-49-6Relevant articles and documents
Mechanism and active site residues of GDP-fucose synthase
Lau, Stephen T. B.,Tanner, Martin E.
experimental part, p. 17593 - 17602 (2009/07/19)
L-Fucose, 6-deoxy-L-galactose, is a key component of many important glycoconjugates including the blood group antigens and the Lewisx ligands. The biosynthesis of GDP-L-fucose begins with the action cof a dehydratase that converts GDP-D-mannose into GDP-4-keto-6-deoxy-mannose. The enzyme GDP-fucose synthase, GFS, (also known as GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, GMER) then converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose. The GFS reaction involves epimerizations at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. This manuscript describes studies that elucidate the order of the epimerization steps and the roles of the active site acid/base residues responsible for the epimerizations. An active site mutant, Cys109Ser, produces GDP-6-deoxy-D-altrose as its major product indicating that C-3 epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. The same mutation results in the appearance of a kinetic isotope effect when [3 - 2H]-GDP-6-deoxy-4-keto- mannose is used as a substrate. This indicates that Cys109 is the base responsible for the deprotonation of the substrate at C-3. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5 position of GDP-fucose in the presence of NADP+. This confirms the order of epimerizations and the role of Cys109. Finally, the inactive His179Gln mutant readily catalyzes the wash-out of deuterium from the C-3 position of [3 - 2H]-GDP-6- deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3 followed by C-5 ) and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps.
