Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

Abstract

ABSTRACT The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase ( Tg PNP) stability and crystallized Tg PNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to Tg PNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for Tg PNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for Tg PNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of Tg PNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of Tg PNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa .