Plasmodium falciparum Purine Nucleoside Phosphorylase

Abstract

Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of PfPNP·Immucillin-H (ImmH)·SO4 reveals a homohexamer with ImmH and SO4 bound at each catalytic site. A solvent-filled cavity close to the 5′-hydroxyl group of ImmH suggested that PfPNP can accept additional functional groups at the 5′-carbon. Assays established 5′-methylthioinosine (MTI) as a substrate for PfPNP. MTI is not found in human metabolism. These properties of PfPNP suggest unusual purine pathways in P. falciparum and provide structural and mechanistic foundations for the design of malaria-specific transition state analogue inhibitors. 5′-Methylthio-Immucillin-H (MT-ImmH) was designed to resemble the transition state of PfPNP and binds to PfPNP and human-PNP with Kd values of 2.7 and 303 nm, respectively, to give a discrimination factor of 112. MT-ImmH is the first inhibitor that favors PfPNP inhibition. The structure of PfPNP·MT-ImmH·SO4 shows that the hydrophobic methylthio group inserts into a hydrophobic region adjacent to the more hydrophilic 5′-hydroxyl binding site of ImmH. The catalytic features of PfPNP indicate a dual cellular function in purine salvage and polyamine metabolism. Combined metabolic functions in a single enzyme strengthen the rationale for targeting PfPNP in anti-malarial action. Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of PfPNP·Immucillin-H (ImmH)·SO4 reveals a homohexamer with ImmH and SO4 bound at each catalytic site. A solvent-filled cavity close to the 5′-hydroxyl group of ImmH suggested that PfPNP can accept additional functional groups at the 5′-carbon. Assays established 5′-methylthioinosine (MTI) as a substrate for PfPNP. MTI is not found in human metabolism. These properties of PfPNP suggest unusual purine pathways in P. falciparum and provide structural and mechanistic foundations for the design of malaria-specific transition state analogue inhibitors. 5′-Methylthio-Immucillin-H (MT-ImmH) was designed to resemble the transition state of PfPNP and binds to PfPNP and human-PNP with Kd values of 2.7 and 303 nm, respectively, to give a discrimination factor of 112. MT-ImmH is the first inhibitor that favors PfPNP inhibition. The structure of PfPNP·MT-ImmH·SO4 shows that the hydrophobic methylthio group inserts into a hydrophobic region adjacent to the more hydrophilic 5′-hydroxyl binding site of ImmH. The catalytic features of PfPNP indicate a dual cellular function in purine salvage and polyamine metabolism. Combined metabolic functions in a single enzyme strengthen the rationale for targeting PfPNP in anti-malarial action. Plasmodium falciparum is a purine auxotroph, and starvation of purines is known to cause purine-less death in cultured cells (1Reyes P. Rathod P.K. Sanchez D.J. Mrema J.E. Rieckmann K.H. Heidrich H.G. Mol. Biochem. Parasitol. 1982; 5: 275-290Google Scholar, 2Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2002; 277: 3226-3231Google Scholar, 3Asahi H. Kanazawa T. Kajihara Y. Takahashi K. Takahasi T. Parasitology. 1996; 113: 19-23Google Scholar). Parasites cultured in human erythrocytes can be killed rapidly by blocking PNP 1The abbreviations used are: PNP, purine nucleoside phosphorylase; PfPNP, P. falciparum PNP; huPNP, human PNP; MTA, 5′-methylthioadenosine; MTI, 5′-methylthioinosine; ADA, adenosine deaminase; ImmH, Immucillin-H; MT-ImmH, 5′-methylthio-Immucillin-H. with Immucillin-H and related Immucillins; however, these agents inhibit both P. falciparum and human PNPs, albeit with higher efficiency for human PNP (2Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2002; 277: 3226-3231Google Scholar, 4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar). The biochemical link between PNP inhibition and purine-less death is the formation of hypoxanthine, previously identified as a primary source of purines for P. falciparum (1Reyes P. Rathod P.K. Sanchez D.J. Mrema J.E. Rieckmann K.H. Heidrich H.G. Mol. Biochem. Parasitol. 1982; 5: 275-290Google Scholar, 3Asahi H. Kanazawa T. Kajihara Y. Takahashi K. Takahasi T. Parasitology. 1996; 113: 19-23Google Scholar). The amino acid sequence of P. falciparum PNP as well as its substrate specificity differs from mammalian and bacterial enzymes (4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar, 5Bzowska A. Kulikowska E. Shugar D. Pharmacol. Ther. 2000; 88: 349-425Google Scholar). We investigated this difference by structural analysis using Immucillin-H as a ligand to stabilize the catalytic site and as a starting point for synthesis of inhibitors more specific to PfPNP. Immucillin-H is effective in causing P. falciparum cell death by purine starvation even in the purine-rich environment of human erythrocytes. Hypoxanthine but not inosine can prevent the effect (2Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Schramm V.L. Kim K. J. Biol. Chem. 2002; 277: 3226-3231Google Scholar). The transition state of PfPNP has oxacarbenium ion character and ImmH binds to PfPNP with a dissociation constant of 860 pm, making it a powerful inhibitor for this target (Refs. 4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar and 6Lewandowicz A. Schramm V.L. Biochemistry. 2004; 43: 1458-1468Google Scholar; Fig. 1). However, ImmH was designed to be a transition state analogue of mammalian PNPs and binds with a constant of 56 pm to human PNP (7Miles R.W. Tyler P.C. Furneaux R.H. Bagdassarian C.K. Schramm V.L. Biochemistry. 1998; 37: 8615-8621Google Scholar). Therefore, addition of this compound to human erythrocytes infected with P. falciparum has two effects, inhibition of human PNP at lower concentrations and inhibition of both human and PfPNPs at higher concentrations. Comparison of the PfPNP amino acid sequence against the data base of all PNPs indicated that this enzyme differs from mammalian and bacterial PNPs (see below). The structure of PfPNP in complex with ImmH and SO4 revealed that the catalytic site has capacity for binding 5′-substituted nucleosides. 5′-Methylthioinosine and inosine are good substrates for PfPNP. We hypothesized that 5′-methylthio-substituted Immucillin would bind preferentially to PfPNP. This hypothesis was used to design, produce, and characterize 5′-methylthio-Immucillin-H (MT-ImmH), a novel transition state analogue inhibitor with high specificity for the P. falciparum PNP. PfPNP—The coding sequence for PfPNP (4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar) was amplified by PCR from P. falciparum 3D7 genomic DNA, placed into the pTrcHis2-TOPO vector, expressed in Escherichia coli TOP 10, and purified to >95% homogeneity by nickel affinity chromatography. ImmH was synthesized by the convergent route (8Evans G.B. Furneaux R.H. Hutchison T.L. Kezer H.S. Morris Jr., P.E. Schramm V.L. Tyler P.C. J. Org. Chem. 2001; 66: 5723-5730Google Scholar). MT-ImmH was synthesized by the desilylation of the previously reported N-tert-butoxycarbonyl-7-O-tert-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-O-isopropylidene-d-allo-heptononitrile followed by methanesulfonylation of the 7-hydroxyl and displacement of the resulting mesylate with thiomethoxide. The resultant compound was converted into MT-Immucillin-H in an analogous fashion to that previously described (9Evans G.B. Furneaux R.H. Gainsford G.J. Schramm V.L. Tyler P.C. Tetrahedron. 2000; 56: 3053-3062Google Scholar, 10Kikagawa K. Iizuka K. Higuchi Y. Hirayama H. Ichino M. J. Med. Chem. 1972; 15: 387-390Google Scholar). Structure and stereochemistry of MT-ImmH was confirmed by mass spectrometry and NMR. Inhibition and Kinetic Studies—Inhibitor dissociation constants for human and PfPNP were based on reaction rate measurements with inosine as substrate and variable inhibitor concentrations (4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar, 7Miles R.W. Tyler P.C. Furneaux R.H. Bagdassarian C.K. Schramm V.L. Biochemistry. 1998; 37: 8615-8621Google Scholar). Reactions were started by addition of PNP to 1 mm inosine in 50 mm KPO4, pH = 7.5, 25 °C containing xanthine oxidase at 60 milliunits/ml. Hypoxanthine was oxidized to uric acid and followed spectrophotometrically at 293 nm (7Miles R.W. Tyler P.C. Furneaux R.H. Bagdassarian C.K. Schramm V.L. Biochemistry. 1998; 37: 8615-8621Google Scholar). Dissociation constants were determined according to the equation v = (kcat × S)/(Km(1 + I/Kd) + S), where v is the steady state reaction rate, kcat is the rate at substrate saturation, S is substrate concentration, Km is the Michaelis constant for inosine, I is inhibitor concentration, and Kd is the equilibrium dissociation constant for the PNP-inhibitor complex. Slow onset inhibition was taken into account for inhibitors with dissociation constants less than 1 nm (7Miles R.W. Tyler P.C. Furneaux R.H. Bagdassarian C.K. Schramm V.L. Biochemistry. 1998; 37: 8615-8621Google Scholar). 5′-Methylthioinosine was produced by the action of P. falciparum adenosine deaminase 2L.-M. Ting, W. Shi, A. Lewandowicz, D. C. Madrid, V. Singh, P. C. Tyler, G. B. Evans, R. H. Furneaux, V. L. Schramm, and K. Kim, unpublished data. on 5′-methylthioadenosine and kinetic constants established as described above. Crystallization and X-ray Methods—The complex of PfPNP·ImmH·SO4 was crystallized with 1.5 equivalents of ImmH using hanging-drop vapor diffusion at 18 °C. Protein (30 mg/ml) with ImmH and 3 mm NaH2PO4 was mixed with an equal volume of reservoir solution containing 1.6 m (NH4)2SO4 and 3% isopropanol (Hampton) and equilibrated against 1.0 ml of the reservoir solution. Diffraction from these crystals was consistent with the orthorhombic space group P212121 (a = 86.55 Å, b = 92.29 Å, c = 239.64 Å) with a hexamer in the asymmetric unit (Vm = 2.66 Å3/Da; 53% solvent). Following a brief transfer to mother liquor containing 20% glycerol and flash-cooling to –178 °C, diffraction data were collected on a RAXIS IV area detector using Cu-Kα x-rays (1.54 Å). The structure was solved by molecular replacement methods using the hexamer of the E. coli PNP·formycin-B·SO4 complex (Protein Data Bank ID code 1A69) as the model (11Mao C. Cook W.J. Zhou M. Koszalka G.W. Krenitsky T.A. Ealick S.C. Structure (Lond.). 1997; 5: 1373-1383Google Scholar) and refined to 2.2-Å resolution as previously described (12Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar, 13Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Google Scholar, 14Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Google Scholar). Other parameters and the coordinates have been submitted to the Protein Data Bank (ID code 1NW4). The complex of PfPNP·MT-ImmH·SO4 was crystallized as described for PfPNP·ImmH·SO4 except that MT-ImmH was used in preincubations. Data from the PfPNP·MT-ImmH·SO4 crystals were collected at 0.98-Å wavelength on an ADSC CCD detector at beam line 19BM at the Argonne National Laboratory. The crystals were flash-frozen after a brief transfer to 20% glycerol. The complex of PfPNP·MT-ImmH·SO4 was refined to 2.0-Å resolution using the PfPNP·ImmH·SO4 structure as the starting model (Table I). The coordinates and refinement statistics have been submitted to the Protein Data Bank (ID code 1Q1G).Table IKinetic properties of human and P. falciparum PNP and sequence homologySubstrateHuman PNPP. falciparum PNPKmkcatkcat/KmKmKcatkcat/Kmμms-1m-1 s-1μms-1m-1 s-1Inosine38 ± 331 ± 58 × 1054.7 ± 0.31.7 ± 0.33.6 × 105Methylthioinosine12 ± 30.2 ± 0.12 × 10416 ± 31.5 ± 0.29.6 × 104Methylthioadenosine<0.02<0.02S-Adenosylhomocysteine<0.02<0.02 Open table in a new tab Structure of PfPNP·ImmH·SO4—PfPNP crystallized in the presence of ImmH and sulfate revealed a monomer structure consisting of a core of 10 β-sheets (Fig. 2). The hexameric subunit structure is similar to E. coli PNP but is different from the trimeric mammaliam enzyme (11Mao C. Cook W.J. Zhou M. Koszalka G.W. Krenitsky T.A. Ealick S.C. Structure (Lond.). 1997; 5: 1373-1383Google Scholar, 15Fedorov A. Shi W. Kicska G. Fedorov E. Tyler P.C. Furneaux R.H. Hanson J.C. Gainsford G.J. Larese J.Z. Schramm V.L. Almo S.C. Biochemistry. 2001; 40: 853-860Google Scholar, 16Bennett E.M. Li C. Allan P.W. Parker W.B. Ealick S.E. J. Biol. Chem. 2003; 278: 47110-47118Google Scholar). Monomers are folded into a single domain in which the 10-stranded β-sheet core forms the back of the catalytic site and is surrounded by nine α-helices that form subunit contacts (Fig. 2, a and b). The PfPNP hexamer is organized as a trimer of dimers and the active site of each monomer (Mr 26,900) is fully occupied by one ImmH and one SO4. The interface between the subunits forming the dimer buries a total of 4020 Å2, while that between adjacent dimers is less extensive and buries a total of 2457 Å2. Two equivalent catalytic sites are located between each pair of dimers and are related by a non-crystallographic 2-fold axis. Catalytic site residues are contributed primarily from the parent subunit except that His7 and Arg45 from the adjacent subunit bind to the 5′-hydroxyl of ImmH and the sulfate ion, respectively (Fig. 3). The 9-deazahypoxanthine base of ImmH is sandwiched between the backbone of β5 and β9 on one side and the side chain of Tyr160 from the other side. Tyr160 also forms two water-mediated hydrogen bonds with O5′ and Asp206. Asp206 interacts with N7 of ImmH and is proposed to be the general acid/base for protonation of N7 of substrate at the transition state. His7 from the neighboring subunit, together with Glu184 and the bound sulfate, orient the iminoribitol of ImmH through interactions with 5′-, 2′-, and 3′-hydroxyls. Only 16 of the 245 amino acids of PfPNP are common to P. falciparum, human, and E. coli PNPs, and only two of these appear to contact Immucillin-H in the catalytic site (4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar). The regions adjacent to the 5′-hydroxyl of bound ImmH differ considerably in mammalian and P. falciparum PNPs (Fig. 3). Sulfate ion binds beneath the inhibitor and its oxygens are fully engaged with two hydrogen bonds between each oxygen and protein residues Ser91, Arg88, Gly23 from the parent subunit, Arg45 from the neighboring subunit, and the 3′-hydroxyl of ImmH. The catalytic site cavity near the 5′-hydroxyl group of ImmH in the PfPNP structure contained an isopropyl alcohol molecule from the solvent and suggested the capacity to accept a methylthio or homocysteine group at this position. The mammalian PNPs are more tightly constrained around the 5′-hydroxyl group and do not have this cavity (Fig. 3). PfPNP is specific for 6-oxypurine rings in its substrates, and nucleosides with adenine rings are neither substrates nor inhibitors for the enzyme, while the E. coli enzyme uses both 6-oxy- and 6-aminopurine nucleoside substrates (4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar, 16Bennett E.M. Li C. Allan P.W. Parker W.B. Ealick S.E. J. Biol. Chem. 2003; 278: 47110-47118Google Scholar). The action of PfPNP on a 5′-substituted nucleoside would therefore require 5′-methylthioinosine or 5′-inosylhomocysteine, compounds unknown to mammalian metabolism. Origin of MT-inosine and Catalytic Site Specificity of PfPNP— Despite the structural similarity to E. coli PNP, the catalytic properties of PfPNP are substantially different from both mammalian and E. coli PNPs (4Kicska G.A. Tyler P.C. Evans G.B. Furneaux R.H. Kim K. Schramm V.L. J. Biol. Chem. 2002; 277: 3219-3225Google Scholar, 5Bzowska A. Kulikowska E. Shugar D. Pharmacol. Ther. 2000; 88: 349-425Google Scholar, 16Bennett E.M. Li C. Allan P.W. Parker W.B. Ealick S.E. J. Biol. Chem. 2003; 278: 47110-47118Google Scholar).2 We tested the hypothesis that 5′-methylthioinosine (MTI) could be formed in P. falciparum from 5′-methylthioadenosine (MTA) by its adenosine deaminase (PfADA). 5′-Methylthioadenosine is a common by-product of polyamine metabolism. We overexpressed PfADA in E. coli and found that MTA and adenosine were equivalent substrates (kcat/Km) for purified recombinant PfADA enzyme.2 Mammalian ADA (bovine) showed no activity for MTA. We used PfADA to produce MTI. Both MTI and inosine are good substrates for PfPNP (Table I). Human PNP is designed for 6-oxypurine nucleosides, since there is no MTI in humans. Human PNP does catalyze the phosphorolysis of MTI but its kcat/Km for inosine is 40 times that for MTI. MTI forms in P. falciparum because P. falciparum ADA converts MTA to MTI and there is no 5′-methylthioadenosine phosphorylase in P. falciparum.2 Therefore, PfPNP exhibits catalytic competence for inosine and MTI substrates (Table I). Both huPNP and PfPNP are specific for 6-oxypurines and this specificity was confirmed with MTA and S-adenosylhomocysteine (Table I). Inosine is central to purine salvage and the steps: MTA → MTI → hypoxanthine also appear to be important in purine salvage for the polyamine pathway in P. falciparum. PNP action is crucial for P. falciparum growth without supplemental hypoxanthine. Therefore PfPNP activity lies at an essential metabolic branchpoint. It generates hypoxanthine from both the purine nucleoside pathways and from polyamine synthesis. Inhibitor Design for PfPNP Based on MT-inosine Specificity— The dual substrate specificity of PfPNP reinforces this enzyme as a target for the design of transition state analogue inhibitors as antimalarials. We hypothesized that MT-ImmH could exhibit increased specificity for PfPNP by mimicking a normal transition state structure of the parasite enzyme. We synthesized MT-ImmH using methods similar to those for ImmH synthesis (see “Experimental Procedures”). MT-ImmH binds better to PfPNP than to human PNP by a factor of 112. In contrast, the discrimination factor for ImmH (Kd human PNP/Kd PfPNP) is 0.065. These discrimination factors establish that the methylthio group of MT-ImmH provides specificity for inhibition of PfPNP relative to huPNP (Fig. 1). The Kd values for ImmH and MT-ImmH are similar at 0.86 and 2.7 nm. Relative to the Km values, these transition state analogue inhibitors bind ∼5000-fold tighter than substrates to PfPNP (Fig. 1). Crystal Structure of PfPNP·MT-ImmH·SO4—The overall fold of PfPNP·ImmH·SO4 and PfPNP·MT-ImmH·SO4 complexes are similar with root mean square deviations < 0.3 Å for all 245 Cα atoms in pairwise comparison. In the active site the hydrogen bond networks for the 9-deazahypoxanthine base and the sulfate ion are also similar with the exception of the water molecule interacting with Asp206 and Tyr160. In PfPNP·ImmH·SO4 the water molecule is in contact with the 5′-hydroxyl group of ImmH, while in PfPNP·MT-ImmH·SO4 this water molecule shifted ∼1 Å and forms a 2.9-Å hydrogen bond with N7 of the 9-deazahypoxanthine base. The 5′-hydroxyl and 5′-methylthio groups of the two transition-state analogues are in different orientations. The 5′-hydroxyl of ImmH points toward a hydrophilic cavity that leads to the surface of the protein. The cavity includes an isopropanol molecule from the crystallization buffer and the O5′ is ∼3.8 Å away from the isopropanol hydroxyl oxygen (Fig. 3). The O5′ of ImmH is within hydrogen bond distance of both N4′ and the side chain of His7 from the neighboring subunit. In PfPNP·MT-ImmH·SO4 the methyl group attached to S5′ of MT-ImmH is rotated ∼135° compared with the 5′-hydroxyl in ImmH and is 4.0 Å away from N4′ of the iminoribitol ring. The side chain of His7 also rotates ∼135° and stacks to the side of the methylthio group. The methylthio group of MT-ImmH points away from the large hydrophilic cavity and into a closed hydrophobic pocket formed by Tyr160, Val66, Met183 from one subunit, and His7 and Val73 from the neighboring subunit. Most of these residues are not conserved in the mammalian PNP with the exception of Met183 (Met219 in huPNP). Orientation of ImmH, SO4, and surrounding atoms in PfPNP compared with MT-ImmH shows the bound isopropanol and the shift in the enzyme-bound water (Fig. 4). Summary and Conclusions—The crystal structure of PfPNP revealed a catalytic site that can accommodate 5′-methylthioinosine and kinetic studies confirmed that MTI is a good substrate for PfPNP. This specificity indicates that PfPNP functions at the intersection of polyamine metabolism and purine salvage in the pathways: polyamines → MTA → MTI → hypoxanthine and inosine → hypoxanthine. MT-Immucillin-H is a of the transition state for phosphorolysis of MTI. It binds more tightly to PfPNP than to human PNP. MT-Immucillin-H is the first known PNP inhibitor with specificity for the enzyme. resolution crystal of the PfPNP revealed for the 5′-hydroxyl of ImmH and the methylthio group of MT-ImmH, to be by the hydrophilic and hydrophobic at the catalytic site.