| dc.description.abstract | Apicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, are obligate intracellular protozoa that are the cause for human disease and the source of economic damage. Plasmodium and Toxoplasma gondii are purine auxotrophs that rely on the host to maintain their metabolic requirements. Targeting purine salvage and recycling is an approach that has been successful in the disruption of DNA replication. The impetus for novel chemotherapeutics are due to the challenge of drug resistant strains, particularly for malaria.;Purine salvage in Plasmodium is streamlined with adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT) representing the major pathway for purine acquisition. PNP is pertinent for Plasmodium viability, and understanding the mechanism of how the enzyme selects substrates will allow for the creation of inhibitors that can withstand mutations. Plasmodium falciparum enzymes PfADA and PfPNP have unique multiple specificity that enable them to act upon methylthiopurines resulting from polyamine synthesis. Thus Plasmodium ADA and PNP function in both purine salvage and purine recycling. Immucillins, powerful picomolar transition state analogues of PNP, are active against cultured Plasmodium falciparum and inhibit all Plasmodium PNPs tested.;We have explored structural features in Plasmodium PNP that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We show that V66I:V73I:Y160F PfPNP mutation has weak binding for malaria specific transition state inhibitor, 5'methylthio-immucillin-H, compared to wildtype PfPNP. The V66I:V73I:Y160F PfPNP structure was solved, showing the now compacted area preventing favorable binding of the 5'methylthio group.;We solved the structure for Toxoplasma PNP, which has 41% sequence identity to PfPNP but does not utilize methythiopurines. TgPNP•ImmH•PO43- complex reveals a contrast to PfPNP binding through weak activity for the DADMe-Immucillins and MT-ImmH, allowing insight into the substrate specificity of closely related phylum organisms. These Apicomplexan PNPs developed divergent substrate utilization, which is not only shown in the 5' groups accepted, but with the bond length between the iminoribitol and deazapurine groups in the transition state structure.;This information will guide the development of chemotherapeutics, through rational drug design, to an alternative means of treatment for both Toxoplasma and Plasmodium PNP. | |
