Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog.
Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.
Chaudhary
Kshitiz
K
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Ting
Li Min
LM
Kim
Kami
K
Roos
David S
DS
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology*
D009711:Nucleotides; D011683:Purine-Nucleoside Phosphorylase