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• W2035592221 abstract "The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-Å resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-Å resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2—C-3 bond of mannose 6-phosphate. The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-Å resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-Å resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2—C-3 bond of mannose 6-phosphate. Phosphoglucose isomerase (PGI, 1The abbreviations used are: PGI, phosphoglucose isomerase; Fru-6-P, fructose 6-phosphate; Glc-6-P, glucose 6-phosphate, PAB, 5-phosphoarabinonate; PaPGI/PMI, phosphoglucose isomerase/phosphomannose isomerase from P. aerophilum; Man-6-P, mannose 6-phosphate; rPGI, rabbit phosphoglucose isomerase; PMI, phosphomannose isomerase; r.m.s., root mean square. 1The abbreviations used are: PGI, phosphoglucose isomerase; Fru-6-P, fructose 6-phosphate; Glc-6-P, glucose 6-phosphate, PAB, 5-phosphoarabinonate; PaPGI/PMI, phosphoglucose isomerase/phosphomannose isomerase from P. aerophilum; Man-6-P, mannose 6-phosphate; rPGI, rabbit phosphoglucose isomerase; PMI, phosphomannose isomerase; r.m.s., root mean square. EC 5.3.1.9) catalyzes the interconversion of d-glucose 6-phosphate to d-fructose 6-phosphate via an aldose-ketose isomerization reaction. This equilibrium reaction is part of glycolysis and gluconeogenesis but also impacts other pathways in sugar metabolism such as the pentose phosphate pathway. The enzyme from bacterial and mammalian sources has been well characterized. Crystal structures show the enzyme to be a tight homodimer in which the two active sites are located at the domain interface and are formed by elements from both subunits (1Sun Y.J. Chou C.C. Chen W.S. Wu R.T. Meng M. Hsiao C.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5412-5417Crossref PubMed Scopus (105) Google Scholar, 2Jeffery C.J. Bahnson B.J. Chien W. Ringe D. Petsko G.A. Biochemistry. 2000; 39: 955-964Crossref PubMed Scopus (108) Google Scholar, 3Read J. Pearce J. Li X. Muirhead H. Chirgwin J. Davies C. J. Mol. Biol. 2001; 309: 447-464Crossref PubMed Scopus (89) Google Scholar). These structures support a catalytic mechanism for isomerization in which a glutamate (e.g. Glu-357 in rabbit PGI) acts as a base catalyst to remove a proton from C-1 or C-2 (depending on the direction of the reaction), forming a cis-enediolate intermediate. Because the open chain forms of its substrates are expected to be present in vivo in trace amounts (4Swenson C.A. Barker R. Biochemistry. 1971; 10: 3151-3154Crossref PubMed Scopus (58) Google Scholar), PGI also catalyzes a ring-opening reaction. This reaction is acid-catalyzed by a histidine (e.g. His-388 in rabbit PGI) (5Lee J.H. Chang K.Z. Patel V. Jeffery C.J. Biochemistry. 2001; 40: 7799-7805Crossref PubMed Scopus (68) Google Scholar, 6Davies C. Muirhead H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 453-465Crossref PubMed Scopus (17) Google Scholar), and a lysine (Lys-518) also appears to assist this reaction by abstracting a proton from C-1 of Glc-6-P (or C-2 of Fru-6-P) (7Solomons J.T. Burns S. Wessner L. Krishnamurthy N. Zimmerly E. Swan M.K. Krings S. Muirhead H. Chirgwin J. Davies C. J. Mol. Biol. 2004; (in press)PubMed Google Scholar). Sequences homologous with PGI cannot be recognized readily within the genomes of Archaea. In some species, such as the euryarchaeons Pyrococcus furiosus and Thermococcus litoralis, PGI activity appears to be catalyzed by a novel enzyme that is structurally and mechanistically distinct from the PGI superfamily (8Verhees C.H. Huynen M.A. Ward D.E. Schiltz E. de Vos W.M. van der Oost J. J. Biol. Chem. 2001; 276: 40926-40932Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Hansen T. Oehlmann M. Schonheit P. J. Bacteriol. 2001; 183: 3428-3435Crossref PubMed Scopus (43) Google Scholar, 10Jeong J.J. Fushinobu S. Ito S. Jeon B.S. Shoun H. Wakagi T. FEBS Lett. 2003; 535: 200-204Crossref PubMed Scopus (25) Google Scholar). Crystal structures of this protein from P. furiosus show it to contain a cupin fold (11Berrisford J.M. Akerboom J. Turnbull A.P. DeGeus D. Sedelnikova S.E. Staton I. McLeod C.W. Verhees C.H. van der Oost J. Rice D.W. Baker P.J. J. Biol. Chem. 2003; 278: 33290-33297Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) at the heart of which is a metal ion that is believed to mediate a hydride shift mechanism of catalysis (12Swan M.K. Solomons J.T. Beeson C.C. Hansen T. Schonheit P. Davies C. J. Biol. Chem. 2003; 278: 47261-47268Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). An interesting facet of this structure is the lack of any obvious amino acids that might catalyze ring opening, leading to suggestions that, in the extremely high temperatures in which P. furiosus thrives, the proportions of sugars in their straight chain form is sufficient to support the demands of metabolism (12Swan M.K. Solomons J.T. Beeson C.C. Hansen T. Schonheit P. Davies C. J. Biol. Chem. 2003; 278: 47261-47268Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In some aerobic crenarchaeota, genes are present in which sequence similarity to some, but not all, of the highly conserved active site motifs of PGI can be detected, suggesting that, unlike the euryarchaeota, these archaeal species may contain PGIs that are distantly related to eubacterial and eukaryotic PGIs (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The proteins from three of these genes, from Aeropyrum pernix, Thermoplasma acidophilum, and Pyrobaculum aerophilum, have been characterized and show PGI activity (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 14Hansen T. Urbanke K. Schonheit P. Extremophiles,. 2004; (in press)Google Scholar). Most interestingly, these enzymes also exhibit phosphomannose isomerase (PMI) activity and can catalyze the interconversion of mannose 6-phosphate (Man-6-P) (the C-2 epimer of Glc-6-P) to Fru-6-P at an equal rate as Glc-6-P to Fru-6-P (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 14Hansen T. Urbanke K. Schonheit P. Extremophiles,. 2004; (in press)Google Scholar) (Fig. 1). The lack of any recognizable pmi gene in these species suggests that this PMI activity may have a function in vivo. Together with homologues from Sulfolobus species, Thermoplasma volcanicum, and Aquifex aeolicus, these enzymes appear to comprise a novel PGI/PMI family within the PGI superfamily (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The mechanism of PMI activity in these PGI/PMIs is interesting because the specificity of “conventional” PGIs for Glc-6-P and Fru-6-P is essentially absolute (15Rose I.A. Adv. Enzymol. Relat. Areas Mol. Biol. 1975; 43: 491-517PubMed Google Scholar). Although the conventional enzyme can interconvert the anomeric forms of Man-6-P (16Rose I.A. O'Connell E.L. Schray K.J. J. Biol. Chem. 1973; 248: 2232-2234Abstract Full Text PDF PubMed Google Scholar), it will not isomerize this substrate to Fru-6-P or epimerize it to Glc-6-P (except at extremely low and nonphysiological rates (17Seeholzer S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1237-1241Crossref PubMed Scopus (31) Google Scholar)). PMI activity within a PGI can be explained by one of two mechanisms (17Seeholzer S.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1237-1241Crossref PubMed Scopus (31) Google Scholar). One mechanism is to use a second base catalyst in the active site and the reaction proceeds via a trans-enediol intermediate. The other mechanism is to rotate the C-2—C-3 substrate bond after proton abstraction from C-2 and prior to proton donation to C-1 (in the Glc-6-P to Fru-6-P direction), and this would presumably require a larger active site to accommodate the rotation. To determine whether these dual PGI/PMIs do belong to the PGI superfamily and to elucidate the structural basis for both enzyme activities, we have determined the structure of PGI/PMI from P. aerophilum in native form at 1.16-Å resolution and in complex with the PGI inhibitor 5-phosphoarabinonate at 1.45-Å resolution. These structures reveal an unexpectedly high degree of similarity with eubacterial and eukaryotic PGIs, but they also show a subtle difference in the active site architecture that may be responsible for the altered specificity. Structure Determination—The crystallization of PGI/PMI from P. aerophilum (PaPGI/PMI) has been described previously (18Swan M.K. Hansen T. Schönheit P. Davies C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1481-1483Crossref PubMed Scopus (4) Google Scholar). Briefly, the crystals belong to space group P21 with cell dimensions a = 55.1 Å, b = 100.8 Å, c = 55.8 Å, and β = 113.2°, and an initial native data set extending to 1.6 Å was reported (18Swan M.K. Hansen T. Schönheit P. Davies C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1481-1483Crossref PubMed Scopus (4) Google Scholar). The crystals were cryo-protected over a period of several hours by passage through a series of mother liquor solutions (25% polyethylene glycol 8000 (w/v) and 0.22 m ammonium sulfate, buffered with 0.1 m Tris-HCl, pH 8.5) each containing increasing amounts of glycerol in 2% increments up to a maximum of 26%. Heavy atom derivatives were prepared by exchanging this solution with an equivalent solution containing a heavy atom salt. Several such compounds were tested. The method for specific iodination of aromatic residues has been described previously (19Sigler P.B. Biochemistry. 1970; 9: 3609-3617Crossref PubMed Scopus (33) Google Scholar). The crystals were then flash-frozen to -180 °C in situ using a cryostream (X-Stream 2000: Rigaku-MSC). Diffraction data were recorded with an RAXIS-IV++ imaging plate system (Rigaku-MSC) mounted on a Rigaku RU-H3R copper rotating anode generator, operating at 50 kV and 100 mA. The x-ray beam was conditioned with Confocal Maxflux™ optics (Osmic, Inc.). For these data sets, the crystal-to-detector distance was 120 mm, and a typical exposure time was 3 min per 1.0° oscillation image. These data were processed using CrystalClear (20Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). Derivatives were identified by calculation of Patterson maps using PHASES (21Furey W. Swaminathan S. Methods Enzymol. 1996; 277: 590-620Crossref Scopus (255) Google Scholar), and phasing calculations at 2.0 Å were performed using autoSHARP (22de la Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar, 23Bricogne G. Vonrhein C. Flensburg C. Schiltz M. Paciorek W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2023-2030Crossref PubMed Scopus (554) Google Scholar) followed by solvent flattening with phase extension to 1.8 Å using SOLOMON (24Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar). Model Building and Refinement—The experimental phases were the starting point for automated model building using the program ARP/wARP (25Perrakis A. Harkiolaki M. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1445-1450Crossref PubMed Scopus (459) Google Scholar, 26Morris R.J. Perrakis A. Lamzin V.S. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2002; 58: 968-975Crossref PubMed Scopus (221) Google Scholar). To obtain the best data set for refinement, new native data were collected at the SER-CAT beamline ID22 at the Advanced Photon Source (Argonne National Laboratory.). These data were acquired on a MAR225 CCD detector with exposure times of 1 s per image, a crystal-to-detector distance of 100 mm, and an oscillation angle of 0.5°. To ensure high redundancy of the data, 360° were collected. Processing was performed using the HKL2000 software package (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). After refinement of the initial model against these data using REFMAC5 (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar), the model was improved manually using XTALVIEW (29McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2021) Google Scholar). Further refinement cycles consisted of the addition of solvent atoms using ARP/wARP, refinement with REFMAC5, and model building with XTAL-VIEW. Refinement of side chain alternative conformations and anisotropic temperature factors was included toward the end of the refinement process. The secondary structure of the final model was calculated using DSSP (30Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12266) Google Scholar). Inhibitor-bound Structure—Crystals were soaked for 5 days in mother liquor solutions containing 26% glycerol and 5 mm of the PGI inhibitor 5-phosphoarabinonate (PAB) (31Chirgwin J.M. Noltmann E.A. J. Biol. Chem. 1975; 250: 7272-7276Abstract Full Text PDF PubMed Google Scholar). Synchrotron data were collected at the SER-CAT beamline in the same way as the high resolution native data. PAB bound at the active site was visualized by refinement of the native model against the data collected from the ligand-soaked crystal, followed by examination of the|Fo| -|Fc difference electron density map. After fitting of the PAB molecule, the structure was refined by using the same protocol used for the native structure and using the same free R assignments as the native data. Structure superimpositions were performed using the CCP4 program LSQKAB (32Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2343) Google Scholar). Coordinates and structure factors for both structures have been deposited with the Protein Data Bank under code 1TZB for native and 1TZC for complex with PAB. Structure Determination—The structure was solved by multiple isomorphous replacement using three derivatives, two gold and one iodine (Table I). Although three iodine peaks were visible in the Patterson map, this derivative had low phasing power and likely contributed little to the final phases. Phasing with SHARP at 1.8-Å resolution produced an experimental electron density map of excellent quality (Fig. 2a) and permitted automated model building of the entire structure except the methionines at each N terminus. After refinement against high resolution data extending to 1.16 Å, the final model has an R factor of 15.0% and Rfree of 16.4%, with excellent stereochemistry (Table II). This model comprises two subunits (of 300 and 301 residues, respectively), 625 water molecules, as well as several molecules of sulfate and glycerol from the crystallization solution. A molecule of sulfate occupies the substrate phosphate-binding site in the active site of each subunit. Only the C-terminal glutamine of subunit B is not visible in the electron density due to apparent flexibility in the vicinity of the C terminus. The N-terminal methionine appears to be absent from the protein because the amino group of Ala-2 forms an electrostatic interaction with a neighboring aspartate, and there is no room for an additional residue. The extremely high resolution of the structure also permitted the modeling of 47 side chains and some main chain regions with alternative conformations: 22 in subunit A and 25 in subunit B. The final 2(|Fo| -|Fc|) electron density map at 1.16-Å resolution is shown in Fig. 2b.Table IStatistics of the phasing using three derivatives in the multiple isomorphous method at 2.0-Å resolution followed by phase extension to 1.8 ÅDerivativeAuBr2AuKBr1Iod4Concentration of soak (mm)1081.8Length of soak24 h4 days10 daysResolution of data2.02.02.5Completeness95.199.186.5RmergeaRmerge=∑hkl∑i|Ii(hkl)−〈I(hkl)〉|/∑hkl∑iIi(hkl).7.08.013.0No. reflections36,17937,12316,798RisobRiso=|FPH−FP|/∑FP|.17.118.43.7Type of dataisoanoisoanoisoanoPhasing powercPhasing power = FH/Er.m.s. (acentric)0.990.491.180.710.110.09RCullisdRCullis=∑|(FPH±FP|−|FH(calc)|/∑|FPH−FP|, where FP, FPH, and FH are the protein, derivative, and heavy atom structure factors, respectively, and Er.m.s. is the residual lack of closure (|FPH − FP| −|FH|). iso, isomorphous data; ano, anomalous data. (acentric)0.830.390.780.311.111.00a Rmerge=∑hkl∑i|Ii(hkl)−〈I(hkl)〉|/∑hkl∑iIi(hkl).b Riso=|FPH−FP|/∑FP|.c Phasing power = FH/Er.m.s.d RCullis=∑|(FPH±FP|−|FH(calc)|/∑|FPH−FP|, where FP, FPH, and FH are the protein, derivative, and heavy atom structure factors, respectively, and Er.m.s. is the residual lack of closure (|FPH − FP| −|FH|). iso, isomorphous data; ano, anomalous data. Open table in a new tab Table IIData collection and refinement statistics of the native structure and the complex with PABData collectionData setNativePABSoak molarity (mm)5Soak time5 daysResolution of data (Å)36.0-1.16 (1.20-1.16)aNumbers in parentheses are for the outer shell of data.36.0-1.45 (1.50-1.45)No. measured reflections1,062,444287,701No. unique reflections168,93897,954Completeness (%)87.7 (61.8)97.8 (94.3)Mean I > σI38.6 (3.3)13.4 (1.6)RmergebWhere Rmerge=∑hkl∑i|Ii(hkl)−〈I(hkl)〉|/∑hkl∑iIi(hkl).7.4 (34.7)7.8 (46.2)RefinementResolution range36.0-1.1636.0-1.45No. water molecules625439R factor (%)15.017.0R work (%)14.916.9Rfree (%)16.519.2r.m.s. deviations from ideal stereochemistryBond lengths (Å)0.0060.008Bond angles (°)1.181.27B factorsMean B factor (main chain) (Å2)11.715.3r.m.s. deviation in main chain B factor (Å2)0.340.43Mean B factor (side chains and waters) (Å2)16.219.8r.m.s. deviation in side chain B factors (Å2)0.781.40Ramachandran plot% residues in most favored region94.694.6% residues in additionally allowed regions5.25.2% residues in generously allowed regions0.20.2% residues in disallowed regions0.00.0a Numbers in parentheses are for the outer shell of data.b Where Rmerge=∑hkl∑i|Ii(hkl)−〈I(hkl)〉|/∑hkl∑iIi(hkl). Open table in a new tab Structure Description—The structure of PaPGI/PMI is a tight dimer of essentially identical subunits; the two subunits superimpose with an r.m.s. deviation of 0.71 Å for all atoms. The structure of one subunit and the dimer are shown in Fig. 3. The subunit comprises two domains, each of which is built around a parallel β sheet, five-stranded in the N-terminal domain and four-stranded in the C-terminal domain. The N terminus is located approximately between the two domains and extends across the face of the N-terminal domain before forming a β hairpin structure (β1 and β2). Thereafter, the N-terminal domain is comprised of alternating αβ segments with α-helices connecting β strands except for the connection between β4 and β5, which is not helical. After α7, the chain crosses over to the C-terminal domain where the same pattern of alternating αβ structure occurs. The C terminus is helical (α15) and is also located between the two domains. The dimer is compact and globular in shape, with no significant extended structural features (Fig. 3b). Two short helical segments, α11 and α12, undergo domain swapping and are an integral part of the opposite subunit. At the dimer interface are numerous ionic interactions that may contribute to the thermostability of the enzyme. Comparison with Conventional PGI—Even though the sequence similarity between them is barely detectable (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), PaPGI/PMI shares a common fold with conventional PGIs. The structure was superimposed with that of rabbit PGI (rPGI) (6Davies C. Muirhead H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 453-465Crossref PubMed Scopus (17) Google Scholar), using an algorithm based on secondary structure matching (33, Krissinel, K., and Henrick, K. (2003) Proceedings of the 5th International Conference on Molecular Structural Biology (Kungl, A. J., and Kungl, P. J., eds) Vienna, September 3-7, 2003, p. 88,Google Scholar), and shows the structural elements that are common to both proteins (Fig. 4). The overall domain structure is the same, but it is immediately obvious that PaPGI/PMI is far smaller and overlaps mostly with the protein core of rabbit PGI. This superimposition permits a structure-based alignment of the PGI sequences from rabbit and P. aerophilum, which shows the relationship between the two proteins more clearly (Fig. 5). A large part of the absent structure in PaPGI/PMI corresponds to the N-terminal end of rabbit PGI, consisting of seven α helices and two β strands, which together form the outer surface of the protein from rabbit. The absence of β1 and β2 (rPGI nomenclature) leaves a parallel four-stranded β sheet in PaPGI/PMI because in rabbit PGI these two strands are anti-parallel and so create a mixed parallel/anti-parallel six-stranded sheet in that enzyme. The C terminus is also shortened; the final helix (α24 in rPGI) is absent, and prior to that, α15 is only half the length of its equivalent in rPGI, α23. The latter helix is important because in conventional PGI it moves toward the active site after ligand binding and contains a lysine (Lys-518) that is critical for catalysis (34Davies C. Muirhead H. Chirgwin J. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 1111-1113Crossref PubMed Scopus (31) Google Scholar). Finally, in rabbit PGI, the structure that forms a “hook” (α20 and α21), and extends to mediate intersubunit contacts in conventional PGIs (6Davies C. Muirhead H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 453-465Crossref PubMed Scopus (17) Google Scholar), has no counterpart in PaPGI/PMI. In addition to these major differences, many of the connections are shorter in PaPGI/PMI.Fig. 5A structure-based sequence alignment of PaPGI/PMI with rPGI. The PGI sequences from P. aerophilum and rabbit are denoted P.a. and O.c. (for Oryctolagus cuniculus), respectively. The secondary structure assignments for each structure are also shown and are color ramped blue-to-red in the N- to C-terminal direction. Residues within the active site are highlighted in red, including the highly conserved residues responsible for catalysis. This figure was produced using the software SecSeq (D. Brodersen, unpublished).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Complex with 5-Phosphoarabinonate—To establish the identity of the active site of PaPGI/PMI, the structure was determined in complex with PAB, a well known inhibitor of PGI activity (31Chirgwin J.M. Noltmann E.A. J. Biol. Chem. 1975; 250: 7272-7276Abstract Full Text PDF PubMed Google Scholar), at 1.45-Å resolution (Table II). PAB bound in an identical manner to both active sites in the dimer, and the view of molecule A is shown in Fig. 6. At one end of the active site, the phosphate group is oriented by three serines (Ser-48, Ser-87, and Ser-89) and one threonine (Thr-92). In the middle of the inhibitor, the C-4 hydroxyl (equivalent to the ring oxygen in the substrate) is within hydrogen-bonding distance of Lys-298 and His-219 (the latter residue belonging to the adjacent subunit in the dimer); the C-3 hydroxyl is contacted by the amide of Gly-47 and the C-2 hydroxyl by the carbonyl group of His-219. In PAB, a carboxylate group replaces the C-1—C-2 region of the substrate such that O-1α is equivalent to C-1 and the carbon at position 1 is equivalent to C-2 of the substrate. Glu-203 is approximately equidistant from both of these atoms, showing that this residue is best placed to abstract and donate protons to the C-1 and C-2 positions of the substrate. This structure permits a direct comparison with that of rabbit PGI in complex with the same inhibitor (35Jeffery C.J. Hardre R. Salmon L. Biochemistry. 2001; 40: 1560-1566Crossref PubMed Scopus (64) Google Scholar). The active sites of the two structures were superimposed by using the coordinates of PAB (Fig. 7). This shows that the majority of amino acids forming the active site are conserved between conventional PGI and PaPGI/PMIs. The cluster of threonines and serines that forms the sugar phosphate-binding site in conventional PGI (3Read J. Pearce J. Li X. Muirhead H. Chirgwin J. Davies C. J. Mol. Biol. 2001; 309: 447-464Crossref PubMed Scopus (89) Google Scholar) is conserved in PaPGI/PMI as Ser-48, Ser-87, and Thr-92 with just a threonine to serine change at position 89. Residues that are important for catalysis in conventional PGI are also conserved in PaPGI/PMI; Glu-357 in rPGI is represented by Glu-203 in PaPGI/PMI, His-388 by His-219, and Lys-518 by Lys-298. There are some differences, however, most notably a proline (Pro-134) in PaPGI/PMI in place of Gly-271, which lead to an alteration in the conformation of β7 to α6 loop in comparison to the same loop in rPGI, and Thr-291 in place of Gln-511. The homology evident between the two active sites confirms PaPGI/PMI as a member of the PGI superfamily (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In addition, the lack of any residue in the vicinity of the carboxylate group of PAB, other than Glu-203, that might act as a base catalyst, shows that the PMI mechanism of this enzyme is unlikely to use a trans-enediol intermediate (discussed below). Conformational Changes Upon Ligand Binding—To determine whether conformational changes occur in PaPGI/PMI in response to the binding of ligands at the active site in the same manner as PGI from eubacterial and eukaryotic sources (e.g. in rabbit PGI (6Davies C. Muirhead H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 453-465Crossref PubMed Scopus (17) Google Scholar, 36Arsenieva D. Jeffery C. J. Mol. Biol. 2002; 323: 77-84Crossref PubMed Scopus (29) Google Scholar)), the native structure and its complex with PAB were superimposed. The r.m.s. deviations calculated between all main chain atoms in the structures is 0.22 Å. Examining the superimposed structures reveals almost no structural differences between the native and PAB-bound structures (Fig. 8). The exception is a slight shift in the C-terminal helix in subunit B, which is due to an improvement in the ordering of this region compared with the wild-type structure. In particular, the C-terminal residue Gln-302 is now visible and hydrogen bonds a water molecule that is close to the PAB inhibitor. Other than this, the positions of all of the residues within the active site region are essentially unchanged. Moreover, given the very close overlap of residues in rabbit PGI and PaPGI/PMI when both complexed to PAB, it is clear that the native state of PaPGI/PMI is equivalent to the ligand-bound “closed” form of rabbit PGI. A major goal of this work was to determine whether PaPGI/PMI belongs to the superfamily of PGI, as suggested by sequence similarity with some of the motifs that comprise conventional PGI (13Hansen T. Wendorff D. Schonheit P. J. Biol. Chem. 2004; 279: 2262-2272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 18Swan M.K. Hansen T. Schönheit P. Davies C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1481-1" @default.
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