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, Tyr179 and Glu157, 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.
Highlights
6-Deoxysugars, prominently represented by the ubiquitous Lfucose,[1] are functionally important constituents of complex glycans and glycosylated natural products
The GDP-mannose is initially oxidized at C4′′ by a NADP+
We sought to clarify through study of the human GMD (UniProt accession ID: O60547), whether and if so to what extent Gerlt and Gassman’s minimum catalytic principle for β-elimination[31] was incorporated by an actual sugar 1,4-dehydratase that has emerged from evolution through natural selection
Summary
Gerlt and Gassman’s mechanism (Figure 1b) built into hGMD implies a 4,5-enolization of GDP-4′′-keto-mannose under concerted general acid−general base catalysis from Tyr[179] and Glu[157], respectively.[37] In both enzyme structures reporting on the Michaelis complex (Figure 2b,f), the Glu[157] is hydrogen bonded to the C6′′ hydroxy group. Molecular dynamics simulations of enzyme complex with NADPH and the enol (GDP-mannos-4′′,5′′-ene) intermediate show that in 21% of 150 structure snapshots analyzed over a total runtime of 15 ns, the Glu[157] approaches the C5′′ at a distance (∼3.5 Å) plausible for catalytic proton transfer at this position (Figure 3a, for details, see Figures S12 and S13 as well as Table S2).[38] In the remainder time of the simulation, the Glu[157] is in contact with the C6′′ hydroxy group.
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