dTDP-glucose 4,6 dehydratase (dehydratase) catalyzes the formation of dTDP 4-keto, 6-deoxy glucose from dTDP-glucose. Dehydratase utilizes an irreversibly bound NAD+ coenzyme to initially oxidize substrate, dTDP-glucose, then, following water elimination, the intermediate is reduced by the coenzyme, yielding product. In the three-dimensional dehydratase structure, the NAD+ nicotinamide group is bracketed by two trios of highly conserved amino acids. The group on the si face of nicotinamide contains the consensus sequence for the extended short chain dehydrogenase superfamily, enzymes which use NAD+ to catalyze oxidoreductase reactions. The group on the re face of nicotinamide forms a structural motif similar to the catalytic triad in cysteine proteases. The roles of these two groups of residues in the regulation of NAD+ reactivity will be determined. Dehydratase variants will be constructed and will be characterized using an array of techniques. NMR analysis of 15N- and 13C-enriched NAD+ molecules will reveal the microelectronic environment of the nicotinamide in each variant. The microscopic rate constants for all steps in the wild type and variant mechanisms will be determined to understand how deletion of the individual residues affects the kinetics and thermodynamics of each step. Finally, the variants will be crystallized for structural analysis by our collaborator.
