@article {2140,
	title = {Mechanism and Active Site Residues of GDP-Fucose Synthase},
	journal = {Journal of the American Chemical Society},
	volume = {130},
	number = {51},
	year = {2008},
	note = {ISI Document Delivery No.: 406UOTimes Cited: 3Cited Reference Count: 37Lau, Stephen T. B. Tanner, Martin E.},
	month = {Dec},
	pages = {17593-17602},
	type = {Article},
	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 GDPfucose 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 {\textquoteright}{\textquoteright} and C-5 {\textquoteright}{\textquoteright} 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 {\textquoteright}{\textquoteright} 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 {\textquoteright}{\textquoteright}-H-2]-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 {\textquoteright}{\textquoteright}. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5 {\textquoteright}{\textquoteright} 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 {\textquoteright}{\textquoteright} position of [3 {\textquoteright}{\textquoteright}-H-2]-GDP-6-deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3 {\textquoteright}{\textquoteright} followed by C-5 {\textquoteright}{\textquoteright}) and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps.},
	keywords = {6-DEHYDRATASE, ARABIDOPSIS-THALIANA, BIOLOGICAL FUNCTION, CATALYTIC MECHANISM, DTDP-GLUCOSE 4, ESCHERICHIA-COLI, GDP-4-KETO-6-DEOXY-D-MANNOSE EPIMERASE/REDUCTASE, HELICOBACTER-PYLORI, LEUKOCYTE ADHESION, MANNOSE 4, UDP-GALACTOSE 4-EPIMERASE},
	isbn = {0002-7863},
	url = {<Go to ISI>://000263320600076},
	author = {Lau, S. T. B. and Tanner, M. E.}
}
