Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine nucleosides (and 2'-deoxynucleosides) to the free base and ribose-1-phosphate (2-deoxyribose-1-phosphate). The enzyme is found in most procaryotic and eucaryotic tissues but shows considerable variation in physical and chemical properties. In particular, human PNP is a trimer of 97kD and is specific for guanosine, inosine and other 6- oxopurine analogs while E. coli PNP is a hexamer of 150kD and takes adenosine, in addition to guanosine and inosine, as a substrate. The overall goal of this NCDDG is to exploit the differences in human and E. coli PNP to achieve bystander killing in tumors for which some cells have been transfected with the E. coli PNP gene. This laboratory component will investigate the three-dimensional structures of human, bovine and E. coli PNP in order to optimize the selectivity of clinical agents. The structure of human PNP (and the nearly identical bovine PNP) has already been determined using X-ray crystallography. We have crystallized E. coli PNP and will determine its high resolution structure using X-ray crystallography. We will also determine the structure of various complexes of PNP with substrates, analogs and inhibitors. Crystals of both bovine and E. coli PNP diffract to at least 1.5 Angstroms resolution at the Cornell High Energy Synchrotron Source (CHESS). Once both structures are known, we will use the comparison of the active sites and molecular modeling to design nucleoside analogs that prefer the E. coli PNP. In addition, we will use the three-dimensional structure to design E. coli PNP mutants with greater selectivity and efficiency for clinically useful nucleoside analogs.
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