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, Tyr14, Glu161, and Glu165 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 Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 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.

Biosynthesis of the glycosaminoglycan precursor UDP-α-D-glucuronic acid occurs through a 2-fold oxidation of UDP-α-D-glucose that is catalysed by UGDH (UDP-α-D-glucose 6-dehydrogenase). Structure-function relationships for UGDH and proposals for the enzymatic reaction mechanism are reviewed in the present paper, and structure-based sequence comparison is used for subclassification of UGDH family members. The eukaryotic group of enzymes (UGDH-II) utilize an extended C-terminal domain for the formation of complex homohexameric assemblies. The comparably simpler oligomerization behaviour of the prokaryotic group of enzymes (UGDH-I), in which dimeric forms prevail, is traced back to the lack of relevant intersubunit contacts and trimmings within the C-terminal region. The active site of UGDH contains a highly conserved cysteine residue, which plays a key role in covalent catalysis. Elevated glycosaminoglycan formation is implicated in a variety of human diseases, including the progression of tumours. The inhibition of synthesis of UDP-α-D-glucuronic acid using UGDH antagonists might therefore be a useful strategy for therapy.

Background: Human UDP-xylose synthase (hUXS1) is responsible for conversion of UDP-glucuronic acid to UDP-xylose.Results: Crystal structure, molecular dynamics simulations, and reaction course analysis give conclusive insight into the enzymatic mechanism in three catalytic steps.Conclusion: Distortion of sugar pyranose ring in bound substrate facilitates enzymatic reaction.Significance: A detailed mechanism for catalysis by hUXS1 is proposed.