Active site conformational dynamics in human uridine phosphorylase 1.
Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 A resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an "induced-fit" association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.
Roosild
Tarmo P
TP
Department of Drug Development, Nevada Cancer Institute, Las Vegas, Nevada, United States of America. troosild@nvcancer.org
Castronovo
Samantha
S
D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019281:Dimerization; D005472:Fluorouracil / Q000378:metabolism; D006801:Humans; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism; D014543:Uridine Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D014543:Uridine Phosphorylase; D005472:Fluorouracil; D014529:Uridine