Inhibiting hexamer disassembly of human UDP-glucose dehydrogenase by photoactivated amino acid crosslinking

Authors

George Grady, University of Nebraska-Lincoln
Ashley Thelen, University of Nebraska-Lincoln
Jaleen Albers, University of Nebraska-Lincoln
Tong Ju, University of Nebraska-Lincoln
Jiantao Guo, University of Nebraska-LincolnFollow
Joseph J. Barycki, University of Nebraska-LincolnFollow
Melanie A. Simpson, University of Nebraska-LincolnFollow

Date of this Version

2016

Citation

Biochemistry. 2016 June 7; 55(22): 3157–3164.

Comments

Copyright © 2016 American Chemical Society. Used by permission.

Abstract

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD⁺-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength UV in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% tetramer and higher molecular mass species. Activity of the crosslinked enzyme was reduced by almost 60% relative to the uncrosslinked UGDH mutant, and UV exposure had no effect on activity of the wildtype enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.