Research & Teaching Faculty

Default Header Image

Mechanism and Active Site Residues of GDP-Fucose Synthase

TitleMechanism and Active Site Residues of GDP-Fucose Synthase
Publication TypeJournal Article
Year of Publication2008
AuthorsLau, STB, Tanner, ME
JournalJournal of the American Chemical Society
Volume130
Pagination17593-17602
Date PublishedDec
Type of ArticleArticle
ISBN Number0002-7863
Keywords6-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
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 ’’ 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 ’’-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 ’’. 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 ’’-H-2]-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.

URL<Go to ISI>://000263320600076