Eukaryot Cell. 2014 Feb 28. [Epub ahead of print]

INHIBITION AND STRUCTURE OF TOXOPLASMA GONDII PURINE NUCLEOSIDE PHOSPHORYLASE

Donaldson TM¹, Cassera MB, Ho MC, Zhan C, Merino EF, Evans GB, Tyler PC, Almo SC, Schramm VL, Kim K.

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 (TgPNP) stability and crystallized TgPNP with phosphate and Immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of eleven immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared with PfPNP, large substituents surrounding the 5' -hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5' groups is consistent with the inability of TgPNP to catalyze the phosphorylysis of 5' -methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2' -hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. 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.

PMID:

24585883

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