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  • Title: Mechanism and active site residues of GDP-fucose synthase.
    Author: Lau ST, Tanner ME.
    Journal: J Am Chem Soc; 2008 Dec 24; 130(51):17593-602. PubMed ID: 19053199.
    Abstract:
    L-fucose, 6-deoxy-L-galactose, is a key component of many important glycoconjugates including the blood group antigens and the Lewis(X) ligands. The biosynthesis of GDP-L-fucose begins with the action of 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''-(2)H]-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''-(2)H]-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.
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