Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Abstract

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 Å 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.

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

“…However, the fact that the protein was able to adopt a supportive geometry for metal coordination in this key region provides further corroboration of its inherent conformational flexibility. Also notable is that the loop at the back of hUPP2’s active site (residues 282–290) subtly closes around the smaller uracil molecule, relative to the bulkier BAU, exactly as extensively described for hUPP1 (Roosild and Castronovo, 2010). …”

“…The position of the inhibitor and binding site for inorganic phosphate proximate to the dimer interface are indicated. For this figure, the right-side monomers of the dimeric enzymes (blue, turquoise, gold) were least-squares aligned producing slightly greater deviations in the backbone traces of the partnering chains, consistent with inherent interdomain flexibility within these proteins (Roosild et al, 2009; Roosild and Castronovo, 2010). However, substantial variation in the main chain conformation is limited to a single, surface-exposed loop on one face of the enzyme (grey highlight).…”

“…Only recently have multiple structures of the human enzyme, hUPP1 (Roosild et al, 2009; Roosild and Castronovo, 2010), its bovine homologue, bUPP1 (Paul et al, 2010), and a UPP from the parasitic protozoa, Trypanosoma brucei (Larson et al, 2010), revealed key differences between prokaryotic and eukaryotic variations of this enzyme. Most strikingly, eukaryotic enzymes have dimeric biological assemblies while prokaryotic proteins are hexameric.…”

A six‑gene support vector machine classifier contributes to the diagnosis of pediatric septic shock

“…UPP1 functions in the homeostatic regulation of intracellular uridine concentrations and the activation of fluoropyrimidine nucleoside chemotherapeutic agents (42). Uridine displays anti-inflammatory action during lung inflammation (43).…”

Pathway Analysis for Drug Repositioning Based on Public Database Mining

“…Some identified pathways even involve multiple proteins with physical interaction with a drug. For example, TYMS is the primary target of fluorouracil; Uridine Phosphorylase 1 (UPP1) is also an enzyme catalyzing the metabolism of fluorouracil and its physical binding with this drug is supported by the crystal structure of UPP1 bound with fluorouracil (PDB ID: 3NBQ) retrieved from MMDB 120 . Both targets participated in the retrieved pathway of Pyrimidine Metabolism (BSID: 82946) that is responsible for the anti-neoplastic effect of fluorouracil (Table 2 and Supporting Information, Table S4).…”