The CDP-tyvelose 2-epimerase from Thermodesulfatator atlanticus (TaTyvE) catalyzes the C-2 epimerization of CDP-glucose to CDP-mannose.The enzyme uses NAD-dependent oxidation and reduction to achieve C-2 configurational inversion of the substrate.Here, we report the 2.60- crystal structure of the tetrameric TaTyvE with NAD + bound in all subunits and CDP bound in only one subunit (PDB: 9RL0).Comparison of structures shows that upon binding of CDP, the loop of residues Gly197-Trp207 becomes ordered to close over the binding pocket of the sugar substrate.The enzyme active site of the ternary complex is structurally well preorganized and only moderately affected by the induced-fit conformational change.Combined evidence of molecular dynamics simulations and site-directed mutagenesis supports the suggestion that TaTyvE employs a dual catalytic architecture to control substrate specificity.Asn125, within the TNK segment (Thr124-Asn125-Lys126), promotes sampling of catalytically plausible glucose conformations, whereas the VAM segment (Val83-Ala84-Met85) permits broader conformational flexibility for mannose through backbone contacts.These findings are consistent with a modest ~12-fold reduction in activity upon substituting Gln205 with alanine, which provides the only side chain contact with mannose outside the VAM segment.In contrast, no activity was detected for the N125A variant.Substrate analogues featuring deoxygenation, stereo-inversion or fluorination at the C-4 were synthesized to examine the role of the sugar C4-OH.MD simulations indicated that the C4-OH interacts with the Val83 backbone and the side chains of Asn125 and Gln205.Wild-type activity was lost completely with CDP-4-deoxy-glucose and was recovered minimally with CDP-4-fluoroglucose.Additional removal of the hydroxymethyl substituent at the C5 to restrict substrate positioning flexibility resulted in an enhanced reaction rate compared to the parent sugar.Overall, these results highlight the important interplay of structural preorganization and conformational flexibility in TaTyvE for enzyme activity and specificity in the C-2 epimerization of CDP-glucose.
Rapp et al. (Sun,) studied this question.