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• W2018554028 abstract "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 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. 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 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. The phylum Apicomplexa consists of >5000 species of obligate intracellular parasites and is responsible for many important diseases in humans and other animals. Malaria (caused by Plasmodium) is a serious global problem with mortality rates in excess of 1 million a year (1Greenwood B. Mutabingwa T. Nature. 2002; 415: 670-672Crossref PubMed Scopus (516) Google Scholar). Toxoplasma gondii is a chronic infection estimated to affect ∼30% of the world's population and poses a significant threat to immunocompromised individuals and congenitally infected children (2Hill D. Dubey J.P. Clin. Microbiol. Infect. 2002; 8: 634-640Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar). The emergence of drug-resistant malaria parasites and complications associated with long-term treatment of chronic toxoplasmosis underscore the need for new chemotherapeutic agents. Focusing on differences between host and parasite metabolism provides an attractive strategy for identifying potential drug targets. One metabolic discrepancy between apicomplexan parasites and their mammalian hosts is the lack of de novo purine biosynthesis in the former, making them completely reliant on host cells for these essential nutrients (3Berens R.L. Krug E.C. Marr J.J. Marr J.J. Muller M. Biochemistry and Molecular Biology of Parasites. Academic Press, London1995: 323-336Crossref Google Scholar). Apicomplexan purine salvage pathways have been explored using a combination of biochemical, genetic, and genomic studies (4Krug E.C. Marr J.J. Berens R.L. J. Biol. Chem. 1989; 264: 10601-10607Abstract Full Text PDF PubMed Google Scholar, 5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 6Chaudhary K. Donald R.G. Nishi M. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 2005; 280: 22053-22059Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 7Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Crossref PubMed Scopus (53) Google Scholar, 8Chaudhary K. Darling J.A. Fohl L.M. Sullivan Jr., W.J. Donald R.G. Pfefferkorn E.R. Ullman B. Roos D.S. J. Biol. Chem. 2004; 279: 31221-31227Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9Striepen B. Pruijssers A.J. Huang J. Li C. Gubbels M.J. Umejiego N.N. Hedstrom L. Kissinger J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3154-3159Crossref PubMed Scopus (171) Google Scholar, 10Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 11Ting L.M. Shi W. Lewandowicz A. Singh V. Mwakingwe A. Birck M.R. Taylor Ringia E.A. Bench G. Madrid D.C. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2005; 280: 9547-9554Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), providing complete transport and metabolic maps for several species (8Chaudhary K. Darling J.A. Fohl L.M. Sullivan Jr., W.J. Donald R.G. Pfefferkorn E.R. Ullman B. Roos D.S. J. Biol. Chem. 2004; 279: 31221-31227Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Comparative analysis reveals two alternative, and functionally redundant, salvage routes for purine assimilation by Eimeria and Toxoplasma. Adenosine kinase (AK) 3The abbreviations used are: AK, adenosine kinase; AK-, AK knockout T. gondii parasites; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyl transferase; Imm, immucillin; MTA, 5′-methylthio-adenosine; MTAP, methylthioadenosine phosphorylase; MTI, 5′-methylthio-inosine; MT-ImmH, 5′-methylthio-immucillin-H; PNP, purine nucleoside phosphorylase; RACE, rapid amplification of cDNA ends; TgPNP, T. gondii; PfPNP, P. falciparum; EcPNP, E. coli PNP.3The abbreviations used are: AK, adenosine kinase; AK-, AK knockout T. gondii parasites; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyl transferase; Imm, immucillin; MTA, 5′-methylthio-adenosine; MTAP, methylthioadenosine phosphorylase; MTI, 5′-methylthio-inosine; MT-ImmH, 5′-methylthio-immucillin-H; PNP, purine nucleoside phosphorylase; RACE, rapid amplification of cDNA ends; TgPNP, T. gondii; PfPNP, P. falciparum; EcPNP, E. coli PNP. converts the nucleoside adenosine into the nucleotide AMP, and hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) converts guanylate nucleobases into nucleotides, including GMP (see “Discussion”). Cryptosporidium and Theileria rely on AK alone, while only HXGPRT is present in Plasmodium, but the ability to enzymatically inter-convert AMP and IMP provides all of these parasites with a supply of all necessary purine nucleotides. Purine nucleoside phosphorylase (PNP) converts inosine to hypoxanthine and guanosine to guanine, providing an important source of nucleobases for HXGPRT (3Berens R.L. Krug E.C. Marr J.J. Marr J.J. Muller M. Biochemistry and Molecular Biology of Parasites. Academic Press, London1995: 323-336Crossref Google Scholar). PNPs have been examined in a variety of species (12Pugmire E.J. Ealick S.E. Biochem. J. 2002; 361: 1-25Crossref PubMed Google Scholar) and may be grouped into two main families: trimeric forms (such as the human enzyme) are typically ∼31 kDa and prefer 6-oxopurines (e.g. inosine, guanosine), and hexameric PNPs (such as the Escherichia coli enzyme) are ∼26 kDa and active against both 6-oxopurines and 6-aminopurines (e.g. adenosine). P. falciparum PNP (PfPNP) has been characterized in detail, and although its amino acid sequence is most similar to hexameric PNPs, its substrate is distinct from either family (10Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Shi W. Ting L.M. Kicska G.A. Lewandowicz A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Almo S.C. Schramm V.L. J. Biol. Chem. 2004; 279: 18103-18106Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In particular, PfPNP is able to utilize 5′-methothioinosine (MTI), produced by the action of adenosine deaminase on methothioadenosine (MTA), a byproduct of polyamine metabolism. The involvement of PfPNP in both purine and polyamine pathways makes this enzyme an attractive drug target, and rationally designed PNP inhibitors (immucillins) inhibit both parasite and host erythrocyte enzymes to produce purine-less death of P. falciparum parasites (11Ting L.M. Shi W. Lewandowicz A. Singh V. Mwakingwe A. Birck M.R. Taylor Ringia E.A. Bench G. Madrid D.C. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2005; 280: 9547-9554Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 14Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2002; 277: 3226-3231Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This report describes the cloning, recombinant expression, and characterization of T. gondii PNP (TgPNP) and comparison with PfPNP. Although broadly similar, TgPNP lacks activity against methylthio-purines. Surprisingly, examination of the T. gondii genome (15Kissinger J.C. Gajria B. Li L. Paulsen I.T. Roos D.S. Nucleic Acids Res. 2003; 31: 234-236Crossref PubMed Scopus (156) Google Scholar) indicates the absence of polyamine biosynthetic machinery in this parasite. Parasites, Host Cells, Chemicals, and Reagents—RH strain T. gondii tachyzoites and adenosine kinase knock-out (AK-) mutants (7Sullivan Jr., W.J. Chiang C.W. Wilson C.M. Naguib F.N. el Kouni M.H. Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 1-14Crossref PubMed Scopus (53) Google Scholar) were maintained by serial passage in primary human foreskin fibroblasts (16Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (497) Google Scholar). Immucillins were generously provided by Drs. Peter C. Tyler, Gary B. Evans, and Richard H. Furneaux (Industrial Research Ltd., Lower Hutt, New Zealand), and Dr. Vern L. Schramm (Albert Einstein College of Medicine, Bronx NY). [3H]uracil (20 Ci mmol-1) was purchased from Moravek Biochemicals (Brea, CA). Xanthine oxidase and all purine substrates were obtained from Sigma. DNA-modifying enzymes were obtained from New England Biolabs (Ipswich, MA). Sensitivity of T. gondii to ImmH and Rescue by Purine Nucleobases—The growth of intracellular T. gondii (wild-type and AK- mutants) was measured in 24-well plates containing confluent human foreskin fibroblast cell monolayers by the incorporation of [3H]uracil into acid-precipitable material. Parasites were grown for 24 h in the presence of varying concentrations of ImmH (0-100 μm), after which the cultures were subjected to a 4-h pulse of [3H]uracil (5 μCi; 20 Ci mmol-1) and plates were processed as previously described (16Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (497) Google Scholar). For metabolic rescue experiments, AK- parasites were inoculated into 96-well plates containing 10 μm ImmH plus various concentrations of hypoxanthine or xanthine (0-100 μm) for 4 days. The disruption of host cell monolayers (an indicator of parasite viability) was measured by crystal violet staining and optical density measurements at 650 nm (16Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (497) Google Scholar). Genomic Analysis and Cloning of T. gondii Nucleoside Phosphorylases—T. gondii genome sequence data (10-fold coverage) are available at //ToxoDB.org (15Kissinger J.C. Gajria B. Li L. Paulsen I.T. Roos D.S. Nucleic Acids Res. 2003; 31: 234-236Crossref PubMed Scopus (156) Google Scholar), and TBLASTN (WU-BLAST 2.0) (17Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68351) Google Scholar) was used to search predicted protein sequences for similarity to the PNP, uridine phosphorylase (UdP), methylthio-adenosine phosphorylase (MTAP), methylthio-adenosine nucleosidase, and polyamine biosynthetic pathway enzymes from various organisms. Based on the most significant match in the T. gondii genome obtained when PNP sequences were used as query, the following primers were constructed for 5′- and 3′-RACE (rapid amplification of cDNA ends) using the SMART™ RACE cDNA amplification kit (Clontech, Palo Alto CA): 5′-GCTGCCCGGGTACTTCGATCGCC-3′ (sense primer for 3′-RACE); 5′-GGCACAGACCGAGGACACCGGAC-3′ (antisense primer for 5′-RACE). T. gondii tachyzoite cDNA was synthesized from total cellular RNA prepared using the RNeasy RNA extraction kit (Qiagen, Valencia CA), and the complete TgPNP open reading frame was amplified using sense primer 5′-acatgcATGCAGGGCATGGAAGTTCAGCCTC-3′ and antisense primer 5′-cgggatccGTACTGGCGACGCAGATTC-3′ (uppercase indicates native coding sequence; restriction sites underlined). The PCR product was gel purified (Qiagen Gel Extraction kit), digested with SphI and BglII, ligated into appropriately digested pQE-70 plasmid (Qiagen), and its sequence verified. The resulting construct (pQE-TgPNP-His6) encodes TgPNP in-frame with a C-terminal His6tagunderthecontrolofanisopropyl-1-thio-β-d-galactopyranoside-inducible promoter. A second putative nucleoside phosphorylase was also identified and cloned from T. gondii (see supplemental materials) and has been shown to function as a UdP (not shown). Nucleoside phosphorylase amino acid sequences obtained from GenBank™ were aligned with T. gondii nucleoside phosphorylases (TgPNP and TgUdP) using ClustalX (18Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35074) Google Scholar). Unambiguously aligned sequences were used to construct phylogenetic trees using the neighbor-joining method (19Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar) and subjected to bootstrap analysis with 1000 replicates (20Hillis D. Bull J. Syst. Biol. 1993; 42: 182-192Crossref Scopus (3630) Google Scholar). Expression and Purification of TgPNP—E. coli strain M15[pREP4] (Qiagen) was transformed with the pQE-TgPNP-His6 plasmid and grown at 37 °C in 100 μg/ml ampicillin and 25 μg/ml kanamycin. Expression of His-tagged protein was induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside when the culture A600 reached 0.6, and cells were harvested 5 h later by centrifugation at 4000 × g for 20 min. Isopropyl-1-thio-β-d-galactopyranoside-induced, TgPNP-transformed E. coli cells were resuspended and sonicated in lysis buffer (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole, 1 mg ml-1 lysozyme) containing a protease inhibitor mixture (Sigma). The lysate was then cleared by centrifugation at 10,000 × g for 20 min. 1 ml of nickel-nitrilotriacetic acid-agarose (Qiagen) was added to the cleared lysate, mixed gently for 1 h, packed into a column, and washed twice with 4 ml of wash buffer (50 mm NaH2PO4, 300 mm NaCl, 30 mm imidazole). Tagged protein was then released with elution buffer (50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole) and estimated to be >95% pure based on denaturing polyacrylamide gel electrophoresis and staining with Coomassie Blue. Protein concentration was measured using a protein assay kit (Bio-Rad, Hercules, CA). Enzyme Assays—All PNP assays were performed with purified enzyme in 50 mm KPO4, pH 7.4. Phosphorylysis of inosine, 2-deoxyinosine, and 5′-methylthio-inosine was measured in a coupled assay with 115 milliunits ml-1 xanthine oxidase to convert hypoxanthine into uric acid. Uric acid formation was followed by spectrophotometric measurement at 293 nm (E293 = 12.9 mm-1 cm-1). Guanosine phosphorylysis was monitored by measuring the disappearance of guanosine at 258 nm (E258 = 5.2 mm-1 cm-1). Adenosine and 5′-methylthio-adenosine phosphorylase activities were measured by following the disappearance of the substrate at 274 nm (E256 = 1.9 mm-1 cm-1). Uridine phosphorylase activity was measured by following the conversion of uridine to uracil at 272 nm (E260 = 2.9 mm-1 cm-1). For inhibition assays, excess substrate (0.5 mm) was used in combination with inhibitor ranging from 0 to 10 μm; the concentration of inhibitor was always at least 10-fold greater than the enzyme concentration. The rapidly reversible inhibition of PNP was analyzed by fitting to the equation v0 = (kcat × S)/(Km (1 + I/Ki) + S), where v0 is the initial reaction rate, kcat the maximal catalytic rate, S the substrate concentration, Km the Michaelis constant, I the inhibitor concentration, and Ki the dissociation constant for the enzyme-inhibitor complex. Because transition state mimics typically exhibit a slow-onset tight binding inhibition (21Schramm V.L. Annu. Rev. Biochem. 1998; 67: 693-720Crossref PubMed Scopus (235) Google Scholar), reaction rates were measured continuously to monitor for a second phase with a markedly different steady state rate vs, in which case Ki*(the dissociation constant for steady state following slow-onset inhibition) was assessed by fitting to vs = (kcat × S)/(Km (1+I/Ki*) + S). Immucillin-H Inhibits the Growth of AK- Parasites but Not Wild-type T. gondii—Immucillins are transition state inhibitors that exhibit potent activity against mammalian and P. falciparum PNPs (14Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2002; 277: 3226-3231Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 22Miles R.W. Tyler P.C. Furneaux R.H. Bagdassarian C.K. Schramm V.L. Biochemistry. 1998; 37: 8615-8621Crossref PubMed Scopus (250) Google Scholar). ImmH kills P. falciparum in culture with an IC50 of 35 nm, with the mode of action dependent on inhibition of both Plasmodium and erythrocyte PNP (14Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2002; 277: 3226-3231Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In contrast, wild-type RH strain T. gondii parasites (WT) are insensitive to 100 μm ImmH (Fig. 1A). Parasites lacking the purine salvage enzyme adenosine kinase (AK-) exhibit a dose-dependent inhibition of growth, however, with an IC50 value of 23 ± 9 nm. (Inhibition of host cell PNP is also expected at this concentration, as ImmH inhibits human PNP with a Ki of 72 pm.) The difference in sensitivities between wild-type and AK- parasites is consistent with previous observations on the redundancy of purine salvage pathways in T. gondii (8Chaudhary K. Darling J.A. Fohl L.M. Sullivan Jr., W.J. Donald R.G. Pfefferkorn E.R. Ullman B. Roos D.S. J. Biol. Chem. 2004; 279: 31221-31227Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar): this parasite can survive by salvaging either adenosine (via AK) or hypoxanthine (via HXGPRT). AK- parasites are dependent on HXGPRT, which may depend on PNP activity as a source of nucleobases. To determine whether ImmH exerts its effect by targeting T. gondii PNP (as opposed to the host enzyme or another target), we assayed the ability of hypoxanthine and xanthine to rescue drug-treated AK- parasites, as shown in Fig. 1B. Both of these nucleobases were able to rescue AK- parasites from the inhibitory effects of 10 μm ImmH. For example, 25 μm hypoxanthine restored parasite viability to 75% of the levels observed without ImmH treatment, and 25 μm xanthine restored parasite viability to 60% of control levels. In contrast, incubation with up to 100 μm concentrations of the nucleoside inosine was unable to rescue AK- parasites from the effects of ImmH (not shown). These data strongly suggest that ImmH acts by inhibiting T. gondii PNP directly. Identification of T. gondii PNP—Similarity searches using sequences from other species to interrogate the T. gondii genome data base identified a single significant match to the P. falciparum (10Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and E. coli (23Mao C. Cook W.J. Zhou M. Koszalka G.W. Krenitsky T.A. Ealick S.E. Structure. 1997; 5: 1373-1383Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) PNP genes, but not to human PNP (24Williams S.R. Goddard J.M. Martin Jr., D.W. Nucleic Acids Res. 1984; 12: 5779-5787Crossref PubMed Scopus (64) Google Scholar). Further analysis of protein ortholog groups (25Li L. Stoeckert Jr., C.J. Roos D.S. Genome Res. 2003; 13: 2178-2190Crossref PubMed Scopus (3947) Google Scholar) also identified another putative nucleoside phosphorylase in the T. gondii genome based on significant similarity to E. coli uridine phosphorylase (26Morgunova E. Mikhailov A.M. Popov A.N. Blagova E.V. Smirnova E.A. Vainshtein B.K. Mao C. Armstrong Sh R. Ealick S.E. Komissarov A.A. Linkova E.V. Burlakova A.A. Mironov A.S. Debabov V.G. FEBS Lett. 1995; 367: 183-187Crossref PubMed Scopus (57) Google Scholar) (see supplemental Fig. S1). Preliminary enzymatic characterization (not shown) indicates that this gene exhibits UdP activity. The full-length cDNA sequence for TgPNP was obtained by 5′- and 3′-RACE and predicts a 247-amino acid protein of molecular mass 26,803 Da. Alignment of TgPNP with related enzymes shows 39% average similarity to the hexameric PNPs (Family 1, including bacterial PNPs and UdPs) versus 21% similarity to trimeric PNPs, (Family 2, including mammalian PNPs and eukaryotic MTAPs). TgPNP exhibits 41% sequence identity to PfPNP and 27% identity to EcPNP (Fig. 2). Phylogenetic analysis also associates the apicomplexan PNPs with Family 1, as TgPNP and PfPNP cluster more closely with UdPs than with any of the PNPs (Fig. 3). TgUdP is an outlier, not grouping strongly with other UdPs or PNPs.FIGURE 3Phylogeny reconstruction for PNP, UdP, and MTAPs. Amino acid sequences that could be unambiguously aligned from several organisms were used to construct a neighbor-joining tree with horizontal branch lengths proportional to distance in the unrooted tree (bar indicates scale in substitutions/site). Solid dots define clades with 100% bootstrap support (shaded dot, 92% support) based on 1000 replicates. HsMTAP, H. sapiens MTAP (GenBank™ accession code Q13126); BstPNPI, B. stearothermophilus PNPI (JT0873); BsuPNPI, Bacillus subtilis PNPI (D69614); MmPNP, Mus musculus PNP (NP_038660); HsPNP, H. sapiens PNP (PHHUPN); TgUdP, T. gondii UdP (ABC94783); VcUdP, Vibrio cholerae UdP (B82249); EcUdP, E. coli UdP (BAB38184); TgPNP, T. gondii PNP (ABC94782); PfPNP, P. falciparum PNP (CAD51497); StPNP, Salmonella typhimurium PNP (NP_463426); EcPNP, E. coli PNP (BAB38766); BstPNPII, B. stearothermophilus PNPII (JT0874); BsuPNPI, B. subtilis PNPII (O34925).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Neither TgPNP nor PfPNP (10Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) exhibits the complete consensus motif for either family of phosphorylases. Sequence alignment (Fig. 2) shows that eight of the sixteen residues known to be involved in substrate binding in the active site of EcPNP (23Mao C. Cook W.J. Zhou M. Koszalka G.W. Krenitsky T.A. Ealick S.E. Structure. 1997; 5: 1373-1383Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) are conserved and five more are conservatively substituted. The remaining three sites are conserved within the Apicomplexa but differ from EcPNP. Asp-206 in PfPNP and Asp-207 in TgPNP probably correspond to Asp-204 in EcPNP, which has been proposed to be the general acid/base for N7 protonation of the substrate purine ring (13Shi W. Ting L.M. Kicska G.A. Lewandowicz A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Almo S.C. Schramm V.L. J. Biol. Chem. 2004; 279: 18103-18106Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Based on biochemical and structural studies, it has been established that PfPNP exhibits activity against MTI, a metabolite not found in humans (11Ting L.M. Shi W. Lewandowicz A. Singh V. Mwakingwe A. Birck M.R. Taylor Ringia E.A. Bench G. Madrid D.C. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2005; 280: 9547-9554Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 13Shi W. Ting L.M. Kicska G.A. Lewandowicz A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Almo S.C. Schramm V.L. J. Biol. Chem. 2004; 279: 18103-18106Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The crystal structure of PfPNP complexed with the transition state analog MT-ImmH reveals that the methylthio group nestles within a hydrophobic pocket formed by Val-66, Tyr-160, and Met-183 from one subunit and His-7 and Val-73 from a neighboring subunit of the PfPNP hexamer. It is interesting to note that despite the overall similarity of apicomplexan PNPs, three conservative substitutions (Ile-68, Ile-75, and Phe-162) render the methylthio binding pocket of TgPNP more similar to EcPNP, which does not possess activity against MTI. 4L. M. Ting and K. Kim, unpublished observations. Expression, Purification, and Biochemical Characterization of TgPNP—Recombinant TgPNP engineered to contain a C-terminal hexahistidine tag was overexpressed and purified from bacteria using a nickel-nitrilotriacetic acid column under native conditions (Fig. 4). On a denaturing polyacrylamide gel, the protein ran as a single band close to its predicted molecular mass of 27 kDa. The protein was stored in elution buffer (see “Materials and Methods”) at 4 °C with minimal loss of activity over a period of 3 months. Among the various substrates tested against recombinant TgPNP, the highest catalytic efficiencies (kcat/Km) were observed for phosphorylysis of inosine (1.98 × 105 mol-1 s-1) and guanosine (3.83 × 105 mol-1 s-1) as shown in Table 1. The kinetic parameters determined for these substrates were most similar to those observed in PfPNP, with a turnover rate ∼10-fold lower than reported for Homo sapiens PNP and EcPNP. Deoxynucleosides, which serve as substrates for mammalian PNPs, showed very poor catalytic efficiency using either parasite PNP, and virtually no activity was detected against adenosine or uridine (substrates for EcPNP and EcUdP, respectively). Although MTI is readily transformed by PfPNP, this purine is not an effective substrate for TgPNP, with a catalytic efficiency <0.5% than that for inosine. MTA is not a substrate for any of these enzymes. Overall, the substrate specificity of TgPNP is distinct from both mammalian and bacterial PNPs and also from the closely related P. falciparum ortholog.TABLE 1Kinetic constants for PNPs from T. gondii, P. falciparum, human erythrocytes, and E. coliSubstrateT. gondiiP. falciparumH. sapiensaValues from Refs. 10, 13E. coliaValues from Refs. 10, 13Kmkcatkcat/KmKmkcatkcat/KmKmkcatkcat/KmKmkcatkcat/Kmμms–1m–1 s–1μms–1m–1 s–1μms–1m–1 s–1μms–1m–1 s–1Inosine13.1 ± 1.22.60 ± 0.021.98 × 1054.71.12.3 × 10540561.4 × 10670881.3 × 106Deoxy-inosine259 ± 610.48 ± 0.011.85 × 103910.899.8 × 103661802311.3 × 106Guanosine9.4 ± 1.63.60 ± 0.023.83 × 1059.42.62.8 × 10512262.3 × 10620593.9 × 106MT-inosine31.9 ± 2.70.0278.46 × 10210.62.62.4 × 104120.21.6 × 104NDbND, not detectable under reaction conditionsAdenosineNDbND, not detectable under reaction conditionsNDbND, not detectable under reaction conditionsMT-adenosineNDbND, not detectable under reaction conditionsNDbND, not detectable under reaction conditionsUridineNDbND, not detectable under reaction conditions1150.097.8 × 102a Values from Refs. 10Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Shi W. Ting L.M. Kicska G.A. Lewandowicz A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Almo S.C. Schramm V.L. J. Biol. Chem. 2004; 279: 18103-18106Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholarb ND, not detectable under reaction conditions Open table in a new tab Inhibition of TgPNP by Immucillins—Immucillins mimic" @default.
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• W2018554028 cites W1580003617 @default.
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• W2018554028 cites W2064589373 @default.
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• W2018554028 cites W2086509416 @default.
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