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J. Biol. Chem., Vol. 281, Issue 35, 25652-25658, September 1, 2006

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Toxoplasma gondii Purine Nucleoside Phosphorylase Biochemical Characterization, Inhibitor Profiles, and Comparison with the Plasmodium falciparum Ortholog^*

Kshitiz Chaudhary¹, Li Min Ting, Kami Kim, and David S. Roos, An Ellison Medical Foundation Senior Scholar in Global Infectious Diseases²

From the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and Departments of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461

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 µM 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 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.

Received for publication, March 20, 2006 , and in revised form, June 22, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) DQ385445 [GenBank] and DQ385446 [GenBank] .

^* This work was supported in part by research and training grants from the National Institutes of Health and by United States Army Research Grant W81XWH-05-2-0025 (to K. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Present address: Division of Parasitology, New England Biolabs Inc., Ipswich, MA 01938-2723.

² To whom correspondence should be addressed: Dept. of Biology, University of Pennsylvania, 415 S. University Ave., Philadelphia, PA 19104-6018. Tel.: 215-898-2118; Fax: 215-746-6697; E-mail: droos{at}sas.upenn.edu.

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