Elevated production of the matrix
glycosaminoglycan hyaluronan is strongly implicated in epithelial
tumor progression. Inhibition of synthesis of the
hyaluronan precursor
UDP-glucuronic acid (
UDP-GlcUA) therefore presents an emerging target for
cancer therapy. Human
UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two
NAD(+)-dependent steps without release of intermediate
aldehyde, the biosynthetic oxidation of
UDP-glucose (
UDP-Glc) to
UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the
apoenzyme and ternary complexes of the
enzyme bound with
UDP-Glc/
NADH and
UDP-GlcUA/
NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal
NAD(+) binding domain, sequestering substrate and
coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of
coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient
aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore
ligand binding to the
NAD(+) binding domain.