Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Tarmo P Roosild,Samantha Castronovo,Fernando Rodrigues-Lima

doi:10.1371/journal.pone.0012741

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.

Highlights

Uridine phosphorylase (UPP; EC 2.4.2.3) is a ubiquitous enzyme that catalyzes the reversible phosphorolysis of uridine and analogous compounds to uracil and ribose-1-phosphate, playing an important role in pyrimidine salvage and regulation of uridine homeostasis [1,2,3]
Interest in understanding the activity of human uridine phosphorylase stems from its role in the activation of pyrimidine nucleoside analogues used in chemotherapy, such as 5-fluorouracil (5-FU) [6] and its prodrug, capecitabine
Results hUPP1 structure when bound to 5-fluorouracil One of the unexpected findings of the first structures of hUPP1 was the discovery that the domains of the enzymatic dimer are flexibly linked, allowing an interdomain motion from an ‘‘open’’ ligand-free conformation to a ‘‘closed’’ catalytically-active structure [20]

Summary

Introduction

Uridine phosphorylase (UPP; EC 2.4.2.3) is a ubiquitous enzyme that catalyzes the reversible phosphorolysis of uridine and analogous compounds to uracil and ribose-1-phosphate, playing an important role in pyrimidine salvage and regulation of uridine homeostasis [1,2,3]. Interest in understanding the activity of human uridine phosphorylase (hUPP) stems from its role in the activation of pyrimidine nucleoside analogues used in chemotherapy, such as 5-fluorouracil (5-FU) [6] and its prodrug, capecitabine. More recent investigations have explored using hUPP inhibitors to boost cellular uridine concentrations, as a means of limiting the toxic effects of fluoropyrimidine nucleoside exposure to healthy tissues during the course of treatment [9,10] Compounds such as 5benzylacyclouridine (BAU) [11] have been tested for their ability to increase the maximum tolerated dosage and therapeutic index of 5-FU through this uridine-mediated cyto-protective phenomenon [12]

Methods

Results

Conclusion