Structural Determinants of the 5′-Methylthioinosine Specificity of Plasmodium Purine Nucleoside Phosphorylase

Abstract

Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5′-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5′-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5′-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5′-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

“…The subunit interaction loop (Tyr161-Pro167) is disordered and alters the normal hexameric form of PfPNP. An earlier report also showed that mutation of M183A disrupts the oligomeric state of PfPNP to become a poorly active monomer (34).…”

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848

“…An overlay of MMV000848 into either complex revealed that active site residues Asn243 and Glu201 create a polar patch that would provide unfavorable contacts with the hydrophobic carbazole ring of MMV000848. In Pf PNP the corresponding patch is formed by hydrophobic residues Val66 of the main chain and Val73 of the neighboring chain, which is essential to accommodate the methyl-thio groups for Pf PNP ( 18 , 28 , 30 , 34 , 44 ). In addition, the side-chain of Tyr88 would sterically clash with the MMV000848 cyclopentyl ring of MMV000848 ( Fig.…”

“…To date, most efforts in optimizing the specificity of drugs targeting Pf PNP have taken advantage of the dual-specificity of Pf PNP for both purines and methylthiopurines ( 18 , 28 , 30 , 34 , 44 ). This is specific to Plasmodium as PNP from other apicomplexa including from Toxoplasma ( 45 ) are devoid of this dual specificity.…”

Purine and Pyrimidine Pathways as Antimalarial Targets

“…The enzyme responsible for forming purine bases from nucleosides in the purine salvage pathway is PNP, catalyzing the reversible phosphorolysis of inosine to hypoxanthine and ribose 1-phosphate [35]. Plasmodium PNP substrate specificity includes MTI, to generate hypoxanthine and 5-methylthioribose 1-phosphate [36]. MTI appears to be a parasite specific metabolite and has not been identified in mammalian pathways [37].…”