title: Inhibition and structure of Toxoplasma gondii purine nucleoside phosphorylase.
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 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.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA.
authors.forename: Teraya M
authors.initials: TM
authors.lastname: Donaldson
authors.forename: María B
authors.initials: MB
authors.lastname: Cassera
authors.forename: Meng-Chiao
authors.initials: MC
authors.lastname: Ho
authors.forename: Chenyang
authors.initials: C
authors.lastname: Zhan
authors.forename: Emilio F
authors.initials: EF
authors.lastname: Merino
authors.forename: Gary B
authors.initials: GB
authors.lastname: Evans
authors.forename: Peter C
authors.initials: PC
authors.lastname: Tyler
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
authors.forename: Kami
authors.initials: K
authors.lastname: Kim
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011684:Purine Nucleosides / Q000737:chemistry / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011744:Pyrimidinones / Q000737:chemistry; D013379:Substrate Specificity; D014122:Toxoplasma / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011684:Purine Nucleosides; D011744:Pyrimidinones; C113101:forodesine; D011683:Purine-Nucleoside Phosphorylase