A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-dehydratase

Abstract

Biosynthesis of 6-deoxy sugars, including l -fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hexose nucleotide precursors as the first committed step. Here, we determined pre- and postcatalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP-mannose C5″ and C6″, coupled to NADP-mediated hydride transfer from C4″ to C6″. We show that concerted acid–base catalysis from only two active-site groups, Tyr 179 and Glu 157 , promotes a syn 1,4-elimination from an enol (not an enolate) intermediate. We also show that the overall multistep catalytic reaction involves the fewest position changes of enzyme and substrate groups and that it proceeds under conserved exploitation of the basic (minimal) catalytic machinery of short-chain dehydrogenase/reductases.

Fluorinated Mannosides Inhibit Cellular Fucosylation.

“…(a) Endogenous mechanism of action for human GMDS. 21 (b) Natural substrate GDP-mannose (green carbons) was docked at the 4-fluoro-GDP-mannose (G4F) binding site in human GMDS (6GPJ 21 , white carbons) in which also NADP + (cyan carbons) was cocrystalized. (c-i) Unnatural substrate GDP-D-Rha6F2 (c, green carbons) and the proposed metabolic intermediates (d-i, green carbons) were docked in the same active site.…”

Fluorinated Mannosides Inhibit Cellular Fucosylation.

“…Black dotted lines depict expected interactions between atoms leading to a new product, based on the natural mechanism. 21 To test our hypothesized prodrug strategy, we studied the metabolism of Fucotrim I and II to their corresponding GDPanalogs inside cells. To this end, intracellular nucleotide sugar levels were analyzed.…”

“…16 The docking experiments were based on a recent study describing the mechanism of action for human GMDS in detail (Figure 3a). 21 The natural substrate GDPmannose was docked at the 4-fluoro-GDP-mannose (G4F) binding site in a human GMDS crystal structure (6GPJ) in which cofactor NADP + was co-crystallized (Figure 3b). 21 Docking of GDP-D-Rha6F2 in the same binding site resulted in similar poses as for GDP-mannose, suggesting it would still be a substrate for GMDS.…”

“…21 The natural substrate GDPmannose was docked at the 4-fluoro-GDP-mannose (G4F) binding site in a human GMDS crystal structure (6GPJ) in which cofactor NADP + was co-crystallized (Figure 3b). 21 Docking of GDP-D-Rha6F2 in the same binding site resulted in similar poses as for GDP-mannose, suggesting it would still be a substrate for GMDS. The proposed metabolic intermediates (Figure 3d-i) are based on the endogenous mechanism of action for GMDS and a known 4,6-dehydratase inhibitor.…”

“…The proposed metabolic intermediates (Figure 3d-i) are based on the endogenous mechanism of action for GMDS and a known 4,6-dehydratase inhibitor. 16,21 The catalytic residues were positioned such that oxidation (I, Figure 3d), enolization (II, Figure 3e), elimination (III, Figure 3f-g) and reduction (IV, Figure 3h) do not seem to be affected sterically. In the endogenous mechanism enolization would yield the final product.…”