UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

摘要

Background: UDP-glucose dehydrogenase (UGDH) mutants were engineered to perturb hexamer:dimer quaternary structure equilibrium.Results: Dimeric species of UGDH have reduced activity in vitro and in supporting hyaluronan production by cultured cells.Conclusion: Only dynamic UGDH hexamers support robust cellular function.Significance: Manipulation of UGDH activity by hexamer stabilization may offer new therapeutic options in cancer and other pathologies.SUMMARY UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium, and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study, we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132), to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters among the hexameric wild-type enzyme and the engineered mutants revealed structurallydependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium in order to function efficiently and preserve regulated activity in the cell.