Mechanism and Active Site Residues of GDP-Fucose Synthase

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.

Complete 6-Deoxy-d-altro-heptose Biosynthesis Pathway from Campylobacter jejuni

“…Mass spectrometry analysis of reaction products generated in the presence of deuterium was necessary to resolve the issue. Such experiments have been used previously to characterize the C3/C5 epimerization activities of similar enzymes (30,50,51). Our experiments on deuterated reaction compounds clearly demonstrated that Cjj1430 only has C3 epimerization activity on the heptose substrate, which leads to the 4-keto-D-arabino sugar intermediate P4 (Fig.…”

“…Significance of Elucidation of This Pathway-Although C3/C5 epimerases and C3/C5 epimerases/C4 reductases that function on deoxy-hexoses, have attracted much attention for their potential use as antibacterial targets (30,33,34,50,51), no such enzyme involved in heptose modification pathways has been characterized at the biochemical level to date. These enzymes also represent novel antibacterial targets because they are likely involved in the modification of the heptoses found in virulence-associated surface carbohydrates of a variety of pathogens, including several strains of C. jejuni (19,21,22), Campylobacter lari (52), as well as Burkholderia species (53).…”

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

“…The enzymes studied are unique as they use heptose-based substrates, whereas all other C3/C5 epimerases and C3/C5 epimerases/C4 reductases studied to date use hexose-based substrates (2,6,7,14,34). Also the ring configuration and the nucleotide portion of the substrates used by these enzymes differ widely compared with the substrates of previously studied enzymes.…”

“…For example, GFS, GMER, and GME use the epimerization substrate GDP-6-deoxy-4-keto-D-lyxo-mannose that is closely related to the GDP-6-deoxy-4-keto-D-lyxo-heptose used by MlghB and DdahB in this study. However, these enzymes differ from MlghB and DdahB in that they all have additional C4 reductase activity, and GME even generates its own 4-keto from GDP-mannose via additional C4 oxidase activity (6,7,14). Sequence-wise, MlghB and DdahB are less related to these GFS, GMER, and GME enzymes, although they use related substrates, than to the RmlC and RfbC enzymes that only have C3/C5 epimerase activities like MlghB and DdahB but use a very different substrate: dTDP-6-deoxy-Dxylo-4-hexulose (ϭdTDP-4-keto-6-deoxy-glucose) (2,34).…”

“…For example, RmlC (34) and GME (14) start with C5 epimerization, whereas GFS starts with C3 epimerization (7). As a result, although most other C3/C5 epimerases only lead to the formation of two products, a mixture of three products was obtained with MlghB.…”