The Structure of NADH in the Enzyme dTDP-d-glucose Dehydratase (RmlB)

The structure of Streptococcus suis serotype type 2 dTDP-d-glucose 4,6-dehydratase (RmlB)
has been determined to 1.5 Å resolution with its nicotinamide coenzyme and substrate analogue dTDP-xylose bound in an abortive complex. During enzyme turnover, NAD+ abstracts a hydride from the C4‘
atom of dTDP-glucose-forming NADH. After elimination of water, hydride is then transferred back to the
C6‘ atom of dTDP-4-keto-5,6-glucosene-regenerating NAD+. Single-crystal spectroscopic studies unambiguously show that the coenzyme has been trapped as NADH in the crystal. Electron density clearly
demonstrates that in contrast to native structures of RmlB where a flat nicotinamide ring is observed, the
dihydropyridine ring of the reduced cofactor in this complex is found as a boat. The si face, from which the
pro-S hydride is transferred, has a concave surface. Ab initio electronic structure calculations demonstrate
that the presence of an internal hydrogen bond, between the amide NH on the nicotinamide ring and one
of the oxygen atoms on a phosphate group, stabilizes this distorted conformation. Additionally, calculations
show that the hydride donor ability of NADH is influenced by the degree of bending in the ring and may be
influenced by an active-site tyrosine residue (Tyr 161). These results demonstrate the ability of dehydratase
enzymes to fine-tune the redox potential of NADH through conformational changes in the nicotinamide
ring.