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TitleInhibition and structure of toxoplasma gondii purine nucleoside phosphorylase
Publication TypeJournal Article
Year of Publication2014
AuthorsDonaldson, T.M., Cassera M.B., Ho M.-C., Zhan C., Merino E.F., Evans G.B., Tyler P.C., Almo S.C., Schramm V.L., and Kim K.
JournalEukaryotic Cell
Pagination572 - 579
Date Published2014
ISSN15359778 (ISSN)
Keywordsantagonists and inhibitors, catalysis, Catalytic Domain, chemistry, Crystallography, X-Ray, enzyme active site, enzyme inhibitor, Enzyme Inhibitors, enzyme specificity, enzymology, forodesine, genetics, Kinetics, metabolism, protozoal protein, Protozoan Proteins, purine nucleoside, Purine nucleoside phosphorylase, Purine Nucleosides, Purine-Nucleoside Phosphorylase, pyrimidinone derivative, Pyrimidinones, Substrate Specificity, Toxoplasma, X ray crystallography
AbstractThe 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 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, 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 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 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. © 2014, American Society for Microbiology. All Rights Reserved.

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