@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.}
}
@article {1127,
	title = {Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+ phosphoenolpyruvate, and N-acetylmannosaminitol},
	journal = {Journal of Biological Chemistry},
	volume = {280},
	number = {5},
	year = {2005},
	note = {ISI Document Delivery No.: 897DJTimes Cited: 19Cited Reference Count: 58},
	month = {Feb},
	pages = {3555-3563},
	type = {Article},
	abstract = {In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an O-18-labeling study that demonstrates the synthase operates via a C-O bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (beta/alpha)(8) barrel (TIM barrel)type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 Angstrom resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the N-acetylmannosamine carbortyl to the addition of PEP.},
	keywords = {3-DEOXY-D-ARABINO-HEPTULOSONATE-7-PHOSPHATE SYNTHASE, 3-DEOXY-D-MANNO-OCTULOSONATE-8-PHOSPHATE SYNTHASE, ACETYLNEURAMINIC ACID, BISUBSTRATE, CAMPYLOBACTER-JEJUNI, CATALYTIC MECHANISM, CELL GLYCOSYLATION, ENZYMATIC SYNTHESIS, ESCHERICHIA-COLI, INHIBITOR, KDO8P SYNTHASE},
	isbn = {0021-9258},
	url = {<Go to ISI>://000226983900053},
	author = {Gunawan, J. and Simard, D. and Gilbert, M. and Lovering, A. L. and Wakarchuk, W. W. and Tanner, M. E. and Strynadka, N. C. J.}
}
