XXXV Reunião Anual da SBBqResumoID:8016


Structural studies of human purine nucleoside phosphorylase in complex with ligands


Gava, L.M.1; Domingues, M.N. 1; Silva, R.G. 2, Basso, L.A. 2; Santos, D.S. 2 and de Azevedo Jr, W.F.2



1 Departamento de Física, IBILCE/UNESP, São José do Rio Preto, SP, Brasil; 2 Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, PUCRS, Porto Alegre, Brasil.


Purine nucleoside phosphorylase (E.C. 2.4.2.1) is a important enzyme of the purine salvage pathway, responsible for the inter-conversion between (deoxy)nucleosides and bases, that may be converted to uric acid for excretion or reused in nucleic acid biosynthesis. This ubiquitous enzyme catalyzes the reversible cleavage, in the presence of inorganic phosphate (Pi), of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the corresponding purine base. The reaction occurs with inversion of configuration, from α–nucleosides to β–ribose 1-phosphate. Interest in human purine nucleoside phosphorylase (HsPNP) has been increased since the discovery that the congenital absence of its activity causes T-cell impairment in human beings, though keeping normal levels of B-cells. Hence, HsPNP has been a potential target for drug development, which could induce immune suppression to treat, for instance, autoimmune diseases, T-cell leukemia and lymphoma and organ transplantation rejection. Moreover, PNP inhibitors can also be used to avoid cleavage of anticancer and antiviral drugs, since many of these drugs mimic natural purine nucleosides, and can thereby be cleaved by PNP before completing their therapeutic role. HsPNP is an attractive target for drug design and it has been submitted to extensive structure-based design. We have obtained the crystallographic structures of HsPNP in complex with 7-deazaguanine, a substrate analogue and in complex with 2-mercapto-4(3H)-quinazolinone, a PNP inhibitor. Recombinant HsPNP was incubated in the presence of the ligands; the hanging drops were equilibrated by vapour diffusion at 20 °C. X-ray diffraction data were collected at 1.4270 Å wavelength using the Synchrotron Radiation Source at LNLS – Campinas/Brazil. X-ray diffraction data for HsPNP:7-deazaguanine were processed to 2.75 Å resolution and for HsPNP:2-mercapto-4(3H)-quinazolinone to 2.7 Å using de program MOSFLM and scaled with the program SCALA. These structures were determined by standard molecular replacement methods with the program AmoRe. Structure refinement was performed using Refmac5 and the positional refinement and manual rebuilding using XtalView. The overall stereochemical quality of the final model was assessed by the program PROCHECK. Analysis of theses structures can provide important information for the structure-based design of new drugs and the improvement of already identified lead compounds.