Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.

Background: UDP-glucose dehydrogenase (UGDH) polymorphisms were identified in a screen of candidate genes for heart valve defects. Results: Two individual mutants fail to rescue cardiac valve defects in UGDH-deleted zebrafish and have reduced stability in vitro. Conclusion: UGDH loss of function mutations result in a subset of human congenital cardiac valve defects caused by reduced enzyme activity during morphogenesis. Significance: Screening these alleles could predict valve defects.

Human UDP‐glucose dehydrogenase (UGDH) is an enzyme that catalyzes the conversion of UDP‐glucose to UDP‐glucuronate by two successive oxidation reactions. Previous studies have highlighted the importance of several conserved active site residues and many of the mechanistic details have been examined. Recent studies indicate that wild‐type UGDH exists as a hexamer, composed of a trimer of catalytically active dimers. Examination of the available UGDH crystal structure suggests that disruption of its oligomeric state may impact its enzymatic activity. To test this hypothesis, we have designed, expressed, and purified two UGDH mutants, E110A and T325A. Both mutants were found primarily in the dimeric state by gel filtration and sedimentation velocity experiments. Importantly, these engineered dimer mutants have kinetic constants comparable to wild‐type enzyme. The E110A and T325A mutants were subsequently used to examine the importance of the hexameric structure using cell culture models. Results of these studies suggest that the intact hexamer is required for optimal UGDH activity.