Structural and functional examination of UDP-glucose dehydrogenase provides insights into development and cancer
University of Nebraska - Lincoln
UDP-glucose dehydrogenase (UGDH) is an enzyme critical for heart valve formation in embryonic development that exists in an unusual equilibrium of dimeric subunits that are reversibly associated in a functional hexamer. The hexameric form is assembled when the substrate and cofactor for the UGDH catalytic reaction are present and our lab has found that this structure can control the activity of the enzyme. UGDH catalyzes two successive NAD+ dependent oxidations of UDP-glucose to yield UDP-glucuronate. The product, UDP-glucuronate, is required for a variety of cellular processes including incorporation into the extracellular matrix glycosaminoglycan hyaluronan and glucuronidation of steroids by UDP-glucuronosyltransferases for elimination. Elevated levels of hyaluronan are strongly implicated in prostate cancer progression, and UDP-glucuronate is essential for regulation of androgen flux in the prostate. The aim of my research is to understand how quaternary assembly of UGDH influences its allosteric regulation. Our lab has designed a set of quaternary assembly mutants, which I have purified and kinetically characterized to probe the effects of oligomeric state on the catalytic activity and stability of UGDH. The obligate dimer mutant displays five-fold reduced activity compared to wild type and the obligate hexamer has negligible activity. This suggests that when the oligomeric state is fixed, catalytic activity is compromised. In addition, wild type UGDH exhibits a sigmoidal shaped kinetic profile, evident of cooperative substrate binding between the subunits, while the dimer mutant shows no evidence of cooperativity. Wild type UGDH is subject to feedback inhibition by the reduced cofactor, NADH, while the dimer mutant is not. Recently, I engineered a photocrosslinkable mutant in which an unnatural amino acid, p-azidophenylalanine, was incorporated into the dimer-dimer interface and can be covalently crosslinked to interacting moieties, including other dimer subunits. Controlling the in vivo production of UDP-glucuronate will allow us to examine its role in prostate cancer progression. Importantly, this research may lead to the development of novel therapeutics for treatment of advanced stage prostate cancer.