Structural Basis for Substrate Specificity of Escherichia coli Purine Nucleoside Phosphorylase
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m304622200
ISSN1083-351X
AutoresEric M. Bennett, Chenglong Li, Paula W. Allan, William B. Parker, S.E. Ealick,
Tópico(s)Adenosine and Purinergic Signaling
ResumoPurine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2′-deoxypurine nucleosides to the free base and ribose (or 2′-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4′- and 5′-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency. Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2′-deoxypurine nucleosides to the free base and ribose (or 2′-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4′- and 5′-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency. Purine nucleoside phosphorylase (PNP) 1The abbreviations used are: PNPpurine nucleoside phosphorylaseAdoadenosineAraA9-β-d-arabinofuranosyladenineF-Ado2 fluoroadenosineF-Ade2-fluoroadenineF-dAdo2-fluoro-2′-deoxyadenosineF-araA9-β-d-arabinofuranosyl-2-fluoroadenineFMBformycin B (8-aza-7-deazainosine)InoinosineMeP6-methylpurineMeP-dR9-β-d-[2-deoxyribofuranosyl]-6-methylpurineMTP-R9-β-d-ribofuranosyl-6-methylthiopurineR 1-Pribose 1-α-d-phosphateTBNtubercidin (7-deazaadenosine)XylA9-β-d-xylofuranosyladenine. reversibly catalyzes the phosphorolytic cleavage of glycosidic bonds to generate ribose 1-phosphate (R 1-P) and a free purine base. PNPs can be divided into two classes, trimeric and hexameric. Trimeric PNPs are specific for 6-oxopurine nucleosides and are found both in higher organisms and in procaryotes. Trimeric PNPs usually have a molecular mass of about 100 kDa. The structures of several trimeric PNPs have been determined (1Mao C. Structure Determination of Purine Nucleoside Phosphorylase from Bovine Spleen and Escherichia coli: Elucidation of Reaction Mechanism. Ph.D. thesis, Cornell University, Ithaca, NY1995Google Scholar, 2Ealick S.E. Rule S.A. Carter D.C. Greenhough T.J. Babu Y.S. Cook W.J. Habash J. Helliwell J.R. Stoeckler J.D. Parks R.E. Bugg C.E. J. Biol. Chem. 1990; 265: 1812-1820Abstract Full Text PDF PubMed Google Scholar, 3Tebbe J. Bzowska A. Wielgus-Kutrowska B. Schroder W. Kazimierczuk Z. Shugar D. Saenger W. Koellner G. J. Mol. Biol. 1999; 294: 1239-1255Crossref PubMed Scopus (62) Google Scholar, 4Shi W. Basso L.A. Tyler P.C. Furneaux R.H. Blanchard J.S. Almo S.C. Schramm V.L. Biochemistry. 2001; 40: 8204-8215Crossref PubMed Scopus (56) Google Scholar), and the catalytic mechanism has been well studied; the cleavage reaction is believed to proceed through an SN1 mechanism with an oxocarbenium nucleoside intermediate (5Kline P.C. Schramm V.L. Biochemistry. 1993; 32: 13212-13219Crossref PubMed Scopus (206) Google Scholar, 6Kline P.C. Schramm V.L. Biochemistry. 1995; 34: 1153-1162Crossref PubMed Scopus (111) Google Scholar, 7Erion M.D. Stoeckler J.D. Guida W.C. Walter R.L. Ealick S.E. Biochemistry. 1997; 36: 11735-11748Crossref PubMed Scopus (140) Google Scholar), with ordered substrate binding, and with product release as the rate-limiting step (8Bzowska A. Biochim. Biophys. Acta. 2002; 1596: 293-317Crossref PubMed Scopus (49) Google Scholar, 9Wielgus-Kutrowska B. Bzowska A. Tebbe J. Koellner G. Shugar D. Biochim. Biophys. Acta. 2002; 1597: 320-324Crossref PubMed Scopus (20) Google Scholar). Hexameric PNPs are found only in lower organisms and have broader substrate specificity, accepting both 6-amino and 6-oxopurine nucleosides. Hexameric PNPs have a molecular mass of about 150 kDa, and two such structures have been determined (10Mao 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 (122) Google Scholar, 11Appleby T.C. Mathews I.I. Porcelli M. Cacciapuoti G. Ealick S.E. J. Biol. Chem. 2001; 276: 39232-39242Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Although the active site residues of trimeric and hexameric PNPs show considerable differences, the mechanisms are thought to be similar, with kinetic isotope effects consistent with an SN1 mechanism (12Stein R.L. Cordes E.H. J. Biol. Chem. 1981; 256: 767-772Abstract Full Text PDF PubMed Google Scholar, 13Lehikoinen P.K. Sinnott M.L. Krenitsky T.A. Biochem. J. 1989; 257: 355-359Crossref PubMed Scopus (21) Google Scholar) (Scheme 1). purine nucleoside phosphorylase adenosine 9-β-d-arabinofuranosyladenine 2 fluoroadenosine 2-fluoroadenine 2-fluoro-2′-deoxyadenosine 9-β-d-arabinofuranosyl-2-fluoroadenine formycin B (8-aza-7-deazainosine) inosine 6-methylpurine 9-β-d-[2-deoxyribofuranosyl]-6-methylpurine 9-β-d-ribofuranosyl-6-methylthiopurine ribose 1-α-d-phosphate tubercidin (7-deazaadenosine) 9-β-d-xylofuranosyladenine. The differences in substrate specificity between the hexameric and trimeric PNPs suggest a strategy for anticancer suicide gene therapy (14Dranoff G. J. Clin. Oncol. 1998; 16: 2548-2556Crossref PubMed Scopus (64) Google Scholar) in which nontoxic nucleoside prodrugs are cleaved to cytotoxic purine analogs (15Sorscher E.J. Peng S. Bebok Z. Allan P.W. Bennett L.L. Parker W.B. Gene Ther. 1994; 1: 233-238PubMed Google Scholar). In this approach, tumor cells transfected with the gene for hexameric Escherichia coli PNP activate the prodrug, whereas normal cells do not. Excellent in vivo activity against tumors that express E. coli PNP (16Martiniello-Wilks R. Garcia-Aragon J. Daja M.M. Russell P. Both G.W. Molloy P.L. Lockett L.J. Russell P.J. Hum. Gene Ther. 1998; 9: 1617-1626Crossref PubMed Scopus (79) Google Scholar, 17Parker W.B. King S.A. Allan P.W. Bennett L.L. Secrist III, J.A. Montgomery J.A. Gilbert K.S. Waud W.R. Wells A.H. Gillespie G.Y. Sorscher E.J. Hum. Gene Ther. 1997; 8: 1637-1644Crossref PubMed Scopus (106) Google Scholar, 18Puhlmann M. Gnant M. Brown C.K. Alexander H.R. Bartlett D.L. Hum. Gene Ther. 1999; 10: 649-657Crossref PubMed Scopus (88) Google Scholar, 19Waud W.R. Gilbert K.S. Parker W.B. Secrist III, J.A. Montgomery J.A. Bennett L.L. Sorscher E.J. Proc. Am. Assoc. Cancer Res. 1998; 39: 512Google Scholar) has been demonstrated with the analogs 9-β-d-[2-deoxyribofuranosyl]-6-methylpurine (MeP-dR), 9-β-d-arabinofuranosyl-2-fluoroadenine (F-araA), and 2-fluoro-2′-deoxyadenosine (F-dAdo), which are not substrates for trimeric PNPs. The toxic purine analogs (6-methylpurine (MeP) and 2-fluoroadenine (F-Ade)) generated from these prodrugs readily diffuse across cell membranes, have high bystander activity not requiring cell-to-cell contact, and can kill the complete cell population even with gene expression in only 0.1-1% of the cells (15Sorscher E.J. Peng S. Bebok Z. Allan P.W. Bennett L.L. Parker W.B. Gene Ther. 1994; 1: 233-238PubMed Google Scholar, 20Hughes B.W. King S.A. Allan P.W. Parker W.B. Sorscher E.J. J. Biol. Chem. 1998; 273: 2322-2328Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Hughes B.W. Wells A.H. Bebok Z. Gadi V.K. Garver R.I. Parker W.B. Sorscher E.J. Cancer Res. 1995; 55: 3339-3345PubMed Google Scholar). In addition, MeP and F-Ade are toxic to both proliferating and nonproliferating tumor cells (22Parker W.B. Allan P.W. Shaddix S.C. Rose L.M. Speegle H.F. Gillespie G.Y. Bennett L.L. Biochem. Pharmacol. 1998; 55: 1673-1681Crossref PubMed Scopus (100) Google Scholar). These characteristics distinguish PNP anticancer gene therapy from the well studied herpes simplex virus thymidine kinase anti-cancer gene therapy strategy (23Rosenberg S.A. Blaese R.M. Brenner M.K. Deisseroth A.B. Ledley F.D. Lotze M.T. Wilson J.M. Nabel G.J. Cornetta K. Economou J.S. Freemen S.M. Riddell S.R. Oldfield E. Gansbacher B. Dunbar C. Walker R.E. Schuening F.G. Roth J.A. Crystal R.G. Welsh M.J. Culver K. Heslop H.E. Simons J. Wilmott R.W. Habib N.A. et al.Hum. Gene Ther. 1999; 10: 3067-3123Crossref PubMed Scopus (31) Google Scholar, 24Beck C. Cayeux S. Lupton S.D. Dorken B. Blankenstein T. Hum. Gene Ther. 1995; 6: 1525-1530Crossref PubMed Scopus (86) Google Scholar, 25Culver K.W. Ram Z. Wallbridge S. Ishii H. Oldfield E.H. Blaese R.M. Science. 1992; 256: 1550-1552Crossref PubMed Scopus (1461) Google Scholar, 26Dennig C.N. Pitts J.D. Blau H.M. Wilson J.M. Molecular and Cellular Biology of Gene Therapy. Keystone Symposia, Snowbird, UT1997Google Scholar, 27Mesnil M. Yamasaki H. Blau H.M. Wilson J.M. Molecular and Cellular Biology of Gene Therapy. Keystone Symposia, Snowbird, UT1997: 67Google Scholar, 28Wygoda M.R. Wilson M.R. Davis M.A. Trosko J.E. Rehemtulla A. Lawrence T.S. Cancer Res. 1997; 57: 1699-1703PubMed Google Scholar, 29Sacco M.G. Benedetti S. Duflotdancer A. Mesnil M. Bagnasco L. Strina D. Fasolo V. Villa A. Macchi P. Faranda S. Vezzoni P. Finocchiaro G. Gene Ther. 1996; 3: 1151-1156PubMed Google Scholar) and suggest that the selective generation of MeP or F-Ade could elicit considerable activity against solid tumors that have a low growth fraction. To optimize PNP anticancer gene therapy, the prodrug should be completely inert with respect to human enzymes and efficiently cleaved only by tumor cells expressing E. coli PNP. But whereas excellent in vivo antitumor activity has been demonstrated with MeP-dR, F-araA, and F-dAdo, these compounds all have dose-limiting toxicities. A better understanding of the structural basis for substrate specificity of the enzyme would allow modification of the prodrugs coupled with enzyme redesign to maintain activity levels while decreasing toxicity. Therefore, we have used crystallography to study the binding of MeP-dR, F-dAdo, and other nucleoside analogs to E. coli PNP. In our previous work (10Mao 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 (122) Google Scholar), the purine binding site was identified in complexes with R 1-P and 6-iodopurine, whereas Koellner et al. determined the structure of complexes with the inhibitors formycin B (FMB; 8-aza-7-deazainosine) (30Koellner G. Luic M. Shugar D. Saenger W. Bzowska A. J. Mol. Biol. 1998; 280: 153-166Crossref PubMed Scopus (81) Google Scholar) and formycin A (8-aza-7-deazaadenosine) (31Koellner G. Bzowska A. Wielgus-Kutrowska B. Luic M. Steiner T. Saenger W. Stepinski J. J. Mol. Biol. 2002; 315: 351-371Crossref PubMed Scopus (69) Google Scholar). We report here the structure of E. coli PNP in association with 10 purine nucleosides (Fig. 1), which vary in both the base (hypoxanthine, 8-aza-7-deazahypoxanthine, adenine, 7-deazaadenine, 2-fluoroadenine, 6-methylpurine, and 6-methylthiopurine) and sugar (ribose, 2′-deoxyribose, arabinose, and xylose) portion of the molecule. We have also performed kinetic studies on the substrates and carried out computer modeling to examine the role of sugar modifications on reactivity. This information, together with knowledge about the structure of mammalian PNPs (32Koellner G. Luic M. Shugar D. Saenger W. Bzowska A. J. Mol. Biol. 1997; 265: 202-216Crossref PubMed Scopus (79) Google Scholar, 33Mao C. Cook W.J. Zhou M. Federov A.A. Almo S.C. Ealick S.E. Biochemistry. 1998; 37: 7135-7146Crossref PubMed Scopus (108) Google Scholar), should aid in the design of more effective prodrugs that can be readily cleaved by E. coli PNP. Furthermore, an understanding of substrate analog binding should allow us to rationally change the enzyme to cleave novel prodrugs that are not cleaved by either human or wild type bacterial PNPs. Crystallization—Purified E. coli PNP was a gift from Dr. George W. Koszalka of Wellcome Research Laboratories. Crystals have been reported in hexagonal (34Cook W.J. Ealick S.E. Krenitsky T.A. Stoeckler J.D. Helliwell J.R. Bugg C.E. J. Biol. Chem. 1985; 260: 12968-12969Abstract Full Text PDF PubMed Google Scholar) and monoclinic (10Mao 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 (122) Google Scholar) forms. Soaks using monoclinic crystals gave poor ligand electron density. Therefore, the hexagonal crystal form (space group P6122 with three monomers designated A, B, and C per asymmetric unit) was used. Crystals were grown by hanging drop vapor diffusion at room temperature. Drops contained 3 μl of 60 mg/ml E. coli PNP and 3 μl of well solution consisting of 1 ml of 30% ammonium sulfate and 50 mm citrate buffer, pH 5.4. Needle-like crystals appeared in about 1 week, with approximate dimensions of 1 mm in length × 0.1 mm in thickness. Typical unit cell dimensions were a = 122.0 Å and c = 242.8 Å with 56% solvent content. X-ray Intensity Measurements—Ligands were soaked into the crystals for 24-36 h, with concentrations ranging from 5 to 50 mm depending on solubility. Crystals were flash frozen at 100 K with liquid nitrogen, using 30% glycerol as a cryoprotectant, and typically diffracted to about 2.2-Å resolution. Data were collected at CHESS stations A1, F1, and F2. Various CCD x-ray detectors were used, including Area Detector Systems Quantum 1 single module and Quantum 4 mosaic CCD detectors and a Princeton Scientific Instruments 2k CCD detector. Exposure times ranged from 30 to 50 s per frame with a frame typically consisting of 1° of rotation. A total of 50-120° of data were collected depending on beam time constraints and the type of detector used. The data were processed using the programs DENZO (35Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) and SCALEPACK (36Otwinowski Z. Proceedings of the CCP4 Study Weekend: Data Collection and Processing 29-30 January. SERC, Daresbury Laboratory, Warrington, UK1993: 56-62Google Scholar). Table I summarizes the data collection statistics.Table IData collection and refinement statisticsAdoAraAF-AdoF-dAdoFMBInoMeP-dRMTP-RTBNXylAResolution (Å)2.52.01.92.32.32.22.32.42.52.1No. of Reflections29,12058,70870,78839,57737,64545,42638,52240,90630,16155,490% Complete77.482.885.784.979.482.881.894.082.089.2I/σaNumbers in parentheses are for the highest resolution shell8.711.78.68.114.812.118.914.610.413.7(2.9)(4.4)(3.1)(3.7)(5.5)(5.5)(8.3)(13.6)(4.4)(4.8)Redundancy2.52.12.61.85.33.72.22.46.62.7Rsym (%)5.44.55.95.87.46.13.65.67.24.6No. of atoms5451545154475411536754485448538653605348No. of waters255443277394125416334331106339Average B (Å2)33.531.324.528.329.533.025.219.730.225.2R-factor (%)17.721.920.019.121.521.517.323.020.718.9R-free (%)23.324.626.424.327.125.220.627.225.921.0r.m.s.br.m.s., root mean square bond (Å)0.0070.0070.0060.0070.0090.0060.0070.0060.0070.007r.m.s. angle (degrees)1.31.31.41.31.31.31.31.31.31.3a Numbers in parentheses are for the highest resolution shellb r.m.s., root mean square Open table in a new tab Structure Determination and Refinement—The structures were solved by molecular replacement using the program X-PLOR (37Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). The E. coli PNP structure from the monoclinic crystal form (Protein Data Bank code 1ECP) was used as a search model. A set of 3-fold related monomers was positioned in the unit cell and used to calculate a difference Fourier map using the PHASES software (38Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar). The structure was refined by simulated annealing with restricted, isotropic individual B-factors. A final round of crystallographic energy minimization and B-factor refinement, including all reflections, was performed using CNS (39Brünger 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. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Starting R-factors ranged from 38 to 42%. Most of the protein backbone required only minor changes. Manual side chain adjustments were performed using the programs CHAIN (40Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar) and O (41Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The loop region from residue 205 to 220 showed varying degrees of disorder, with the best electron density generated using a weighted (2mFo - dFc)exp(αcalc) map based on SIGMAA (42Read R.J. Acta Crystallogr. A. 1986; 42: 140-149Crossref Scopus (2037) Google Scholar) from the CCP4 package (43Collaborative Computational Project 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Some loops could be built at the 0.8 σ level, but others have missing residues in the final model. Temperature factors in this region were much higher than for the rest of the molecule. Models of inosine (Ino), FMB, 7-deazaadenosine (tubercidin (TBN)), 2-fluoroadenosine (F-Ado), 9-β-d-ribofuranosyl-6-methylthiopurine (MTP-R), MeP-dR, and F-dAdo were directly constructed into the corresponding difference electron density. The ligand densities for adenosine (Ado), 9-β-d-arabinofuranosyladenine (AraA), and 9-β-d-xylofuranosyladenine (XylA) were less clear. These maps were improved by 3-fold averaging using the program RAVE (44Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D. 1999; 55: 941-944Crossref PubMed Scopus (156) Google Scholar). Water molecules were included after the ligand model building. The final models contain three protein chains, three ligand molecules, three ions modeled as phosphates, and several hundred water molecules. B-factors for the ligands sometimes varied considerably among monomers of the same complex. The lowest average ligand B-factors within a complex ranged from 18 (F-Ado monomer C) to 76 (AraA monomer C). In 2Fo - Fc maps based on the final model, density connectivity between sugar and base was observed for all monomers of all complexes except AraA and Ado. Refinement statistics are given in Table II.Table IIKm and Vmax values for some compounds with E. coli PNPExperimentKmkcatkcat/KmμMmin-1F-Ado212 ± 1123 ± 1010.2MTP-R39 ± 367 ± 67.4F-dAdo423 ± 9166 ± 517.2MeP-dR2126 ± 20582 ± 2004.6Ado246 ± 7199 ± 244.3Ino296 ± 2240 ± 12.5F-araAaNote that the structure investigated was AraA rather than F-araA2958 ± 4262.5 ± 0.70.0026XylA—b—, no substrate activity at 100 μg/ml for 8 h———a Note that the structure investigated was AraA rather than F-araAb —, no substrate activity at 100 μg/ml for 8 h Open table in a new tab Determination of Km and Vmax—Enzyme activity was measured in 200-μl volumes containing 50 mm potassium phosphate (pH 7.4). The enzyme concentration was adjusted for each compound so that product formation increased linearly with respect to time. The substrate concentrations were near the Km values and in all cases were in great excess of the enzyme concentration (>1000-fold). After incubation for the desired time at 25 °C, the reaction was stopped by boiling, and the precipitated proteins were removed by filtration (0.2 μm). Reverse phase high pressure liquid chromatography (5-μm BDS Hypersil C-18 column, 150 × 4.6 mm; Keystone Scientific Inc., State College, PA) was used to separate products from substrates, which were detected as they eluted from the column by their absorbance at their absorbance maxima. The mobile phase was 1.25-5% acetonitrile (depending on the nucleoside) in 50 mm ammonium dihydrogen phosphate buffer (pH 4.5) at a flow rate of 1 ml/min. The Michaelis-Menten parameters were determined from linear double reciprocal plots of 1/velocity versus 1/concentration of the substrate. The best line was determined by linear regression of at least five datum points (the regression coefficient for each measurement was greater than 0.95), and the Km and Vmax values were determined from the intercept of the x and y axis, respectively. Each number is the mean of at least two separate determinations. After converting Vmax to kcat, the final kinetic parameters are given in Table II. Modeling of Intermediate Formation—To examine the role of sugar pucker in reaching the proposed oxocarbenium intermediate reaction state, computer modeling was carried out on the Ado, AraA, Ino, and XylA ligands using the GB/SA solvation model (45Qiu D. Shenkin P.S. Hollinger F.P. Still W.C. J. Phys. Chem. A. 1997; 101: 3005-3014Crossref Scopus (931) Google Scholar) and AMBER* force field implemented in MacroModel version 7.2 (46Mohamadi F. Richards N.G.J. Guida W.C. Liskamp R. Lipton M. Caufield C. Chang G. Hendrickson T. Still W.C. J. Comput. Chem. 1990; 11: 460-467Crossref Scopus (3933) Google Scholar). Because the bottom of the energy well of the crystal structure appeared to be wide, a weak restraint of 1.0 kJ/mol-Å2 was applied to the substrate to prevent drifting away from the crystallographic position, whereas the protein was restrained at 200 kJ/mol-Å2, and the phosphate was left unrestrained due to software limitations. To mimic the formation of the oxocarbenium intermediate, the force field parameter for the glycosidic bond was adjusted to a bond length 50% greater than normal, and the O-4′-C-1′-N-9 and C-2′-C-1′-N-9 angle parameters were set to 90°. The structures were energy-minimized with these modified parameters to obtain an approximate model for how the ligand moves during the reaction. Modeling of Ribose 1-Phosphate—Because only density for the adenine base was observed in an attempted soak including R 1-P, the DockVision program (47Hart T.N. Read R.J. Proteins. 1992; 13: 206-222Crossref PubMed Scopus (187) Google Scholar) was utilized to position a R 1-P molecule into the E. coli PNP active site. Both the Monte Carlo simulation option and genetic algorithm within DockVision were used, but only the Monte Carlo simulation gave reasonable results. These calculations were performed using the Cornell Theory Center work station cluster. Four Monte Carlo simulations and two genetic algorithm runs were performed with a distance cut-off of 8.0 Å. For the four Monte Carlo simulations, the numbers of trials were set at 10,000, 20,000, 10,000, and 20,000, and dielectric constants were set at 1, 1, 2, and 4, respectively. For genetic algorithm runs, the number of generations was set at 10,000, and the dielectric constant was set at 1 or 2. The results of the genetic algorithm runs were inconsistent with no convergence. The Monte Carlo simulation gave much better results, and the run with the highest dielectric constant resulted in a cluster of 26 R 1-P structures located near the expected binding site. Of these 26 models, the one with the lowest energy had the best hydrogen bonding interactions and is the one described in this paper. Structure of E. coli PNP—The structures of unliganded E. coli PNP (10Mao 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 (122) Google Scholar) and complexes with formycin B (30Koellner G. Luic M. Shugar D. Saenger W. Bzowska A. J. Mol. Biol. 1998; 280: 153-166Crossref PubMed Scopus (81) Google Scholar) and a formycin A analog (31Koellner G. Bzowska A. Wielgus-Kutrowska B. Luic M. Steiner T. Saenger W. Stepinski J. J. Mol. Biol. 2002; 315: 351-371Crossref PubMed Scopus (69) Google Scholar) have been previously reported. The PNP hexamer is a disc 60 Å thick and 100 Å in diameter, with D3 symmetry and a noncrystallographic 3-fold axis in the crystal form utilized for these studies (Fig. 2). The monomers alternate in an up/down fashion around the disc, with three of the active sites near the top and three near the bottom. Each of the six active sites utilizes residues from a pair of 2-fold related monomers. In the present work, the structures of the three crystallographically independent monomers vary slightly within the hexamer and from complex to complex. The largest difference is the conformation of the loop region from residues 205-220, which follows the catalytically important residue Asp204. The other three subunits of the hexamer are generated by crystallographic symmetry. No global differences in domain orientation are observed for the previously reported structures and the 10 complexes reported here. Each monomer consists of a mixed β-sheet core flanked by eight α-helices. The core can be divided into a large eight-stranded β-sheet and a smaller five-stranded β-sheet that pack together to form a distorted β-barrel, an arrangement also found in human PNP (2Ealick S.E. Rule S.A. Carter D.C. Greenhough T.J. Babu Y.S. Cook W.J. Habash J. Helliwell J.R. Stoeckler J.D. Parks R.E. Bugg C.E. J. Biol. Chem. 1990; 265: 1812-1820Abstract Full Text PDF PubMed Google Scholar), bovine PNP (32Koellner G. Luic M. Shugar D. Saenger W. Bzowska A. J. Mol. Biol. 1997; 265: 202-216Crossref PubMed Scopus (79) Google Scholar, 33Mao C. Cook W.J. Zhou M. Federov A.A. Almo S.C. Ealick S.E. Biochemistry. 1998; 37: 7135-7146Crossref PubMed Scopus (108) Google Scholar), and human 5′-deoxy-5′-methylthioadenosine phosphorylase (48Appleby T.C. Erion M.D. Ealick S.E. Structure. 1999; 7: 629-641Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), although there is little sequence homology between the E. coli enzyme and the trimeric class mammalian enzymes (10Mao 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 (122) Google Scholar). Active Site of E. coli PNP—The previously determined structures of E. coli PNP (10Mao 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 (122) Google Scholar, 30Koellner G. Luic M. Shugar D. Saenger W. Bzowska A. J. Mol. Biol. 1998; 280: 153-166Crossref PubMed Scopus (81) Google Scholar, 31Koellner G. Bzowska A. Wielgus-Kutrowska B. Luic M. Steiner T. Saenger W. Stepinski J. J. Mol. Biol. 2002; 315: 351-371Crossref PubMed Scopus (69) Google Scholar) identified the residues involved in substrate binding and catalysis (Fig. 3). The purine binding site consists of Ala156, Phe159, Val178, Met180, Ile206, and Asp204. The first four of these residues form a hydrophobic pocket around the purine base. Phe159 is located between the purine base and the hydrophobic face of the sugar and makes an angle of ∼60° with the plane of the purine ring. Met180 also lies between the purine base and the hydrophobic face of the ribosyl group. It has been proposed that in E. coli PNP, a protonated Asp204 stabilizes the transition state, in which electron density from the weakening bond is transferred to the purine ring, by donating a hydrogen bond to the purine N-7 atom (10Mao 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 (122) Google Scholar). Evidence for protonation of Asp204 comes from fluorescence studies on the inhibitor FMB, which exists in tautomeric equilibrium between N-7-H and N-8-H forms (IUPAC conventions for ring numbering systems vary among bases; for consistency, we use only the purine ring numbering as shown for inosine in Fig. 1). Although the N-8-H form (which could hydrogen-bond to a protonated Asp204) is the minor (∼20%) tautomer in solution (30Koellner G. Luic M. Shugar D. Saenger W. Bzowska A. J. Mol. Biol. 1998; 280: 153-166Crossref PubMed Scopus (81) Google Scholar, 49Kierdaszuk B. Modrak-Wojcik A. Wierzchowski J. Shugar D. Biochim. Biophys. Acta. 2000; 1476: 109-128Crossref PubMed Scopus (46) Google Scholar), fluorescence studies have detected an equilibrium shift in favor of this tautomer upon binding to E. coli PNP (49Kierdaszuk B. Modrak-Wojcik A. Wierzchowski J. Shugar D. Biochim. Biophys. Acta. 2000; 1476: 109-128Crossref PubMed Scopus (46) Google Scholar). Also consistent with the proposed role for Asp204, mutating this residue to Ala results in a ∼100-fold reduction in activity on MeP-dR and F-araA. 2E. M. Bennett, C. Li, P. W. Allan, W. B. Parker, and S. E. Ealick, unpublished data. The ribose binding site consists primarily of interactions with Glu181 and His4# (residues from an adjacent monomer are designated by # throughout). The Glu181 side chain accepts hydrogen bonds from the 2′- and 3′-hydroxyl groups, and His4# accepts a hydrogen bond from the 5′-hydroxyl group. The phosphate group hydrogen bonds with the 3′-hydroxyl and, in some
Referência(s)