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• W2052283610 abstract "Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of histidine biosynthesis. The enzyme is of fundamental biochemical interest, because it catalyzes removal of a non-acidic hydrogen atom in the dehydration reaction. It is also a potential target for development of herbicides. IGPD is a metalloenzyme in which transition metals induce aggregation and are required for catalysis. Addition of 1 equivalent of Mn2+/subunit is shown by analytical ultracentrifugation to induce the formation of 24-mers from trimeric IGPD. Two histidine-rich motifs may participate in metal binding and aggregation. The 2.3-Å crystal structure of metal-free trimeric IGPD from the fungus Filobasidiella neoformans reveals a novel fold containing an internal repeat, apparently the result of gene duplication. The 95-residue α/β half-domain occurs in a few other proteins, including the GHMP kinase superfamily (galacto-homoserine-mevalonate-phosphomevalonate), but duplication to form a compact domain has not been seen elsewhere. Conserved residues cluster at two types of sites in the trimer, each site containing a conserved histidine-rich motif. A model is proposed for the intact, active 24-mer in which all highly conserved residues, including the histidine-rich motifs in both the N- and C-terminal halves of the polypeptide, cluster at a common site between trimers. This site is a candidate for the active site and also for metal binding leading to aggregation of trimers. The structure provides a basis for further studies of enzyme function and mechanism and for development of more potent and specific herbicides. Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of histidine biosynthesis. The enzyme is of fundamental biochemical interest, because it catalyzes removal of a non-acidic hydrogen atom in the dehydration reaction. It is also a potential target for development of herbicides. IGPD is a metalloenzyme in which transition metals induce aggregation and are required for catalysis. Addition of 1 equivalent of Mn2+/subunit is shown by analytical ultracentrifugation to induce the formation of 24-mers from trimeric IGPD. Two histidine-rich motifs may participate in metal binding and aggregation. The 2.3-Å crystal structure of metal-free trimeric IGPD from the fungus Filobasidiella neoformans reveals a novel fold containing an internal repeat, apparently the result of gene duplication. The 95-residue α/β half-domain occurs in a few other proteins, including the GHMP kinase superfamily (galacto-homoserine-mevalonate-phosphomevalonate), but duplication to form a compact domain has not been seen elsewhere. Conserved residues cluster at two types of sites in the trimer, each site containing a conserved histidine-rich motif. A model is proposed for the intact, active 24-mer in which all highly conserved residues, including the histidine-rich motifs in both the N- and C-terminal halves of the polypeptide, cluster at a common site between trimers. This site is a candidate for the active site and also for metal binding leading to aggregation of trimers. The structure provides a basis for further studies of enzyme function and mechanism and for development of more potent and specific herbicides. Histidine is an essential dietary nutrient for animals but is synthesized de novo by plants and microorganisms. Thus, the biosynthetic pathway is a potential target for herbicide development. Histidine biosynthesis from the precursor phosphoribosyl pyrophosphate is a complex process involving nine enzyme-catalyzed steps. Many intermediates in the pathway are unstable, which has slowed the structural and mechanistic study of individual steps. Imidazole glycerol-phosphate dehydratase (IGPD) 1The abbreviations used are: IGPD, imidazole glycerol-phosphate dehydratase; IGP, imidazole glycerol phosphate; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; HTPP, 2-hydroxy-3-(1,2,4-triazol-1-yl)propylphosphonate; IRL-1803, 3-hydroxy-3-(1,2,4-triazol-3-yl)cyclohexylphosphonate; GHMP, galacto-homoserine-mevalonatephosphomevalonate. 1The abbreviations used are: IGPD, imidazole glycerol-phosphate dehydratase; IGP, imidazole glycerol phosphate; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; HTPP, 2-hydroxy-3-(1,2,4-triazol-1-yl)propylphosphonate; IRL-1803, 3-hydroxy-3-(1,2,4-triazol-3-yl)cyclohexylphosphonate; GHMP, galacto-homoserine-mevalonatephosphomevalonate. catalyzes the sixth step in this pathway, the dehydration of imidazole glycerol phosphate (IGP) to imidazole acetol phosphate (1Ames B.N. J. Biol. Chem. 1957; 228: 131-143Abstract Full Text PDF PubMed Google Scholar). Several IGPD inhibitors have been identified, including derivatives of triazole propyl phosphonic acid (2Hawkes T.R. Cox J.M. Barnes N.J. Beautement K. Edwards L.S. Kipps M.R. Langford M.P. Lewis T. Ridley S.M. Thomas P.G. Brighton Crop Protection Conference-Weeds. 2. Brighton, England1993: 739-744Google Scholar, 3Hawkes T.R. Thomas P.G. Edwards L.S. Rayner S.J. Wilkinson K.W. Rice D.W. Biochem. J. 1995; 306: 385-397Crossref PubMed Scopus (19) Google Scholar, 4Mori I. Iwasaki G. Kimura Y. Matsunaga S. Ogawa A. Nakano T. Buser H.-P. Hatano M. Tada S. Hayakawa K. J. Am. Chem. Soc. 1995; 117: 4411-4412Crossref Scopus (33) Google Scholar). Some of the inhibitors have substantial herbicidal activity (5Mori I. Fonne-Pfister R. Matsunaga S. Tada S. Kimura Y. Iwasaki G. Mano J. Hatano M. Nakano T. Koizumi S. Scheidegger A. Hayakawa K. Ohta D. Plant Physiol. 1995; 107: 719-723Crossref PubMed Scopus (52) Google Scholar). The IGPD-catalyzed reaction may have an unusual molecular mechanism because the leaving C-2 hydrogen of IGP is not acidic. In contrast, the leaving hydrogen in most enzyme-catalyzed dehydrations is relatively acidic because of an adjacent carbonyl or imine group (6Gerlt J.A. Gassman P.G. J. Am. Chem. Soc. 1992; 114: 5928-5934Crossref Scopus (216) Google Scholar). IGPDs from fungi (3Hawkes T.R. Thomas P.G. Edwards L.S. Rayner S.J. Wilkinson K.W. Rice D.W. Biochem. J. 1995; 306: 385-397Crossref PubMed Scopus (19) Google Scholar, 7Parker A.R. Moore T.D. Edman J.C. Schwab J.M. Davisson V.J. Gene (Amst.). 1994; 145: 135-138Crossref PubMed Scopus (14) Google Scholar), plants (8Tada S. Volrath S. Guyer D. Scheidegger A. Ryals J. Ohta D. Ward E. Plant Physiol. 1994; 105: 579-583Crossref PubMed Scopus (20) Google Scholar, 9Mano J. Hatano M. Koizumi S. Tada S. Hashimoto M. Scheidegger A. Plant Physiol. 1993; 103: 733-739Crossref PubMed Scopus (16) Google Scholar), archaea, and some eubacteria are monofunctional. Other eubacteria encode bifunctional enzymes, in which IGPD is fused to histidinol-phosphate phosphatase, the penultimate enzyme of histidine biosynthesis (10Loper J.C. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1440-1449Crossref PubMed Scopus (25) Google Scholar, 11Chiariotti L. Nappo A.G. Carlomagno M.S. Bruni C.B. Mol. Gen. Genet. 1986; 202: 42-47Crossref PubMed Scopus (35) Google Scholar, 12Houston L.L. J. Biol. Chem. 1973; 248: 4144-4149Abstract Full Text PDF PubMed Google Scholar). Metal ions are essential to IGPD catalysis (1Ames B.N. J. Biol. Chem. 1957; 228: 131-143Abstract Full Text PDF PubMed Google Scholar), but the role of metal in promoting the reaction is poorly defined. In the absence of metals, plant and fungal IGPDs are stable inactive trimers (3Hawkes T.R. Thomas P.G. Edwards L.S. Rayner S.J. Wilkinson K.W. Rice D.W. Biochem. J. 1995; 306: 385-397Crossref PubMed Scopus (19) Google Scholar, 13Parker A. Medicinal Chemistry and Molecular Pharmacognosy. Purdue University, West Lafayette, IN1995: 218Google Scholar, 14Tada S. Hatano M. Nakayama Y. Volrath S. Guyer D. Ward E. Ohta D. Plant Physiol. 1995; 109: 153-159Crossref PubMed Scopus (30) Google Scholar). Mn2+ induces aggregation to a catalytically competent form that appears to be a 24-mer (3Hawkes T.R. Thomas P.G. Edwards L.S. Rayner S.J. Wilkinson K.W. Rice D.W. Biochem. J. 1995; 306: 385-397Crossref PubMed Scopus (19) Google Scholar, 9Mano J. Hatano M. Koizumi S. Tada S. Hashimoto M. Scheidegger A. Plant Physiol. 1993; 103: 733-739Crossref PubMed Scopus (16) Google Scholar, 13Parker A. Medicinal Chemistry and Molecular Pharmacognosy. Purdue University, West Lafayette, IN1995: 218Google Scholar, 14Tada S. Hatano M. Nakayama Y. Volrath S. Guyer D. Ward E. Ohta D. Plant Physiol. 1995; 109: 153-159Crossref PubMed Scopus (30) Google Scholar, 15Glaser R.D. Houston L.L. Biochemistry. 1974; 13: 5145-5152Crossref PubMed Scopus (16) Google Scholar). Aggregation has confounded attempts to obtain a three-dimensional structure for IGPD. Preliminary studies (16Wilkinson K.W. Baker P.J. Rice D.W. Rodgers H.F. Stillman T.J. Acta Crystallogr. Sect. D. 1995; 51: 845-847Crossref PubMed Scopus (5) Google Scholar) suggested that yeast IGPD crystallized as a 24-mer with molecular octahedral (432) symmetry consistent with its aggregation in solution. However, the cubic crystals diffracted too poorly for structure determination. The his3 gene of the fungus Filobasidiella neoformans (formerly named Cryptococcus neoformans) encodes the 202-residue IGPD polypeptide (7Parker A.R. Moore T.D. Edman J.C. Schwab J.M. Davisson V.J. Gene (Amst.). 1994; 145: 135-138Crossref PubMed Scopus (14) Google Scholar). The sequences of IGPD from F. neoformans and other sources have no detectable relationship with any protein of known structure. Hence, the fold and structural organization of IGPD cannot be inferred by homology. Amino acids essential to catalysis have not been identified, but several conserved histidine, glutamate, and aspartate residues may serve as metal ligands or play roles in catalysis. In particular, two repeats of the motif Asx-Xaa-His-His-Xaa-Xaa-Glu ((D/N)XHHXXE) are potential metal binding sites (7Parker A.R. Moore T.D. Edman J.C. Schwab J.M. Davisson V.J. Gene (Amst.). 1994; 145: 135-138Crossref PubMed Scopus (14) Google Scholar) especially as histidine imidazoles have been implicated in metal binding (17Petersen J. Hawkes T.R. Lowe D.J. J. Inorg. Biochem. 2000; 80: 161-168Crossref PubMed Scopus (13) Google Scholar). Two occurrences of this motif (residues 69-75 and 165-171 in F. neoformans IGPD) are suggestive of an ancient gene duplication event. IGPD is an interesting candidate for structural studies because of the unusual chemical reaction and aggregation properties, the metal dependence of these properties, the lack of sequence similarity to proteins of known structure, and the potential as a herbicide target. We present here the 2.3-Å crystal structure of F. neoformans IGPD. The structure reveals a new fold in which an unusual structural motif is duplicated into a single compact domain. The structure also provides clues about the location of the active site, allows modeling of the active 24-mer, is a basis for elucidating the mechanism of dehydration, and aids the development of more potent and specific herbicides. Purification and Demetallation of F. neoformans IGPD—F. neoformans IGPD was expressed using the his3 expression vector pHIS3-T7 in Escherichia coli strain BL21(DE3)pLysS (7Parker A.R. Moore T.D. Edman J.C. Schwab J.M. Davisson V.J. Gene (Amst.). 1994; 145: 135-138Crossref PubMed Scopus (14) Google Scholar, 13Parker A. Medicinal Chemistry and Molecular Pharmacognosy. Purdue University, West Lafayette, IN1995: 218Google Scholar). One-liter cultures were grown at 37 °C in 2× YT medium, 100 μg/ml ampicillin, 34 μg/ml chloramphenicol to an A550 of 1.0 and for 8 h after induction with 1.5 mm isopropyl-1-thio-β-d-galactopyranoside. Cells were harvested by centrifugation at 4200 × g for 10 min, resuspended in 40 ml of 20 mm EPPS, pH 8.1 (Buffer A), and disrupted by sonication. DNA was removed with 1% (w/v) in streptomycin sulfate. IGPD was purified in three steps by anion exchange chromatography (Q-Sepharose column in Buffer A with a 0-1 m NaCl gradient), precipitation in 25% saturated (NH4)2SO4, and size-exclusion chromatography (Sephacryl S-400 column in Buffer A). Enzymatic activity was assayed by monitoring absorbance of the enol form of imidazole acetol phosphate (3Hawkes T.R. Thomas P.G. Edwards L.S. Rayner S.J. Wilkinson K.W. Rice D.W. Biochem. J. 1995; 306: 385-397Crossref PubMed Scopus (19) Google Scholar). The yield from 1 liter of E. coli culture was 95 mg of purified IGPD with a specific activity of 5.8 μmol/min/μg of IGPD at 30 °C. Purified IGPD was concentrated to 2.8 mg/ml and stored at -80 °C. F. neoformans IGPD was demetallated by a 6-h incubation at 55 °C in Buffer A containing 4 m urea, 400 μm EDTA, 400 μm EGTA, dialyzed for 1 h at room temperature and 14 h at 4 °C against Buffer A containing 3 g/liter Chelex 100, and then concentrated to ∼14 mg/ml (0.5 mm) by vacuum dialysis. IGPD was reconstituted by the addition of MnCl2 to a final concentration of 1 mm then the IGPD was stored at 4 °C and used for crystallization experiments within 48 h. Analytical Ultracentrifugation—Demetallated IGPD was dialyzed against 0.2 m KCl, 50 mm ethanolamine, pH 7.5, which had been pretreated with Chelex 100. For sedimentation velocity experiments, a standard two-sector carbon-filled Epon centerpiece with quartz windows was treated with 0.1 m EDTA, pH 7.5, for 24 h prior to use, washed with Chelex-treated water to remove EDTA, filled with 150 μl of 1 mg/ml metal-free IGPD, and incubated in a Beckman analytical ultracentrifuge (model XLA) for 1 h at 20 °C. Absorbance scans of metal-free IGPD were collected at 5-min intervals during sedimentation at 42,000 rpm. The centrifuge was stopped, 1 equivalent of Mn2+/IGPD monomer was added to the cell, the solution was carefully mixed, and the cell was incubated in the centrifuge for 1 h. Absorbance scans of Mn2+-containing IGPD were collected at 5-min intervals during sedimentation at 22,000 rpm. The resulting scans were analyzed using the continuous distribution (c(s)) analysis module in the program Sedfit v8.7 (analyticalultracentrifugation.com). Partial specific volumes and solvent densities were calculated using the program SEDNTERP v1.07, which was obtained from the RASMB program depository (rasmb.bbri.org/rasmb/spin/ms_dos/sednterp-philo/). Crystallization and Data Collection—IGPD was crystallized by hanging drop vapor diffusion from a 1:1 mixture of protein solution (14 mg/ml IGPD, 1 mm MnCl2, 20 mm HEPPS, pH 8.1) and reservoir solution (0.9 m (NH4)2SO4, 100 mm sodium acetate, pH 5.0). Crystals of cubic morphology grew to an average dimension of 0.2 mm in approximately 1 week. A mercury-derivative crystal was prepared by a 1.25-h soak in reservoir solution containing 1.0 mm ethyl mercury phosphate, followed by 0.5-h back soak in reservoir solution. IGPD was also crystallized with 1-2 mm concentration of two triazole phosphonic acid derivatives, 2-hydroxy-3-(1,2,4-triazol-1-yl)propylphosphonate (HTPP) (2Hawkes T.R. Cox J.M. Barnes N.J. Beautement K. Edwards L.S. Kipps M.R. Langford M.P. Lewis T. Ridley S.M. Thomas P.G. Brighton Crop Protection Conference-Weeds. 2. Brighton, England1993: 739-744Google Scholar) and 3-hydroxy-3-(1,2,4-triazol-3-yl)cyclohexylphosphonate (IRL-1803) (4Mori I. Iwasaki G. Kimura Y. Matsunaga S. Ogawa A. Nakano T. Buser H.-P. Hatano M. Tada S. Hayakawa K. J. Am. Chem. Soc. 1995; 117: 4411-4412Crossref Scopus (33) Google Scholar). Crystals were cryoprotected by successive transfers through reservoir solution with increasing glycerol to a final concentration of 25% (v/v) and flash-frozen in a nitrogen cold stream at 100 K. Multiwavelength anomalous diffraction data at the mercury LIII edge were recorded at BM-14 at the European Synchrotron Radiation Facility using a Mar 345 imaging plate detector. At each wavelength, data were recorded in two 30° sweeps related by inverse geometry. The same regions of reciprocal space were recorded in “high resolution” and “low resolution” passes. Data from native crystals and from IGPD crystals grown with either HTPP or IRL-1803 were recorded on an R-axis IIc imaging plate using CuKα radiation. Data were processed using the HKL package (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-325Crossref Scopus (38436) Google Scholar). IGPD crystallized in the cubic space group P213 with unit cell parameter 105.3 Å, which is consistent with two polypeptides/ asymmetric unit (Vm = 2.2 Å3/Da, ∼42% solvent). Data quality is summarized in Table I.Table IData collection and phasingHg-IGPDFree enzymeDataWavelengthλ1λ2λ3CuKα1.0086 Å1.0078 Å0.9150 Å1.5418 Å12.2928 keV12.3026 keV13.5503 keV8.0416 keVData range (Å)20-2.3 (2.38-2.30)aValues in parentheses pertain to the outermost shell of data.20-2.3 (2.38-2.30)20-2.3 (2.38-2.30)30-2.6 (2.69-2.60)Unique reflections17,38717,46317,32412,103Average multiplicity12.012.210.01.82Completeness (%)99.1 (100.0)99.3 (100.0)98.7 (99.9)95.6 (84.7)Rsym (%)bRsym=∑h,i|Ih,i-〈Ih〉|/∑h,iIh,i.6.4 (26.1)6.9 (35.0)6.6 (21.3)6.9 (33.7)〈I/σI〉16.8 (3.9)15.2 (2.9)17.8 (4.6)15.5 (3.6)MAD phasingdmin (Å)Overall6.714.763.883.363.002.742.542.37No. of refs.17,07710211497184721052370256127422934〈FOM〉c〈FOM〉, figure of merit; average estimated cosine of phase error.0.380.740.680.590.540.420.270.170.10a Values in parentheses pertain to the outermost shell of data.b Rsym=∑h,i|Ih,i-〈Ih〉|/∑h,iIh,i.c 〈FOM〉, figure of merit; average estimated cosine of phase error. Open table in a new tab Multiwavelength Anomalous Diffraction Structure Determination and Refinement—Structure determination was complicated by translational non-crystallographic symmetry along the body diagonal of the cubic unit cell, revealed by a peak in the native Patterson map (∼10% of the origin peak) at u = v = w = 0.47. Two mercury sites were located by inspection of a Patterson map calculated with coefficients|FA|, which were derived from the multiwavelength anomalous diffraction data using the program SOLVE (19Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar). Four additional mercury sites were found using automated procedures in SOLVE. The final figure of merit was 0.38 for six mercury sites (Table I). Ten cycles of phase refinement via 2-fold averaging, solvent flattening, and histogram matching in the program DM (20Cowtan K.D. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar) yielded an interpretable 2.37-Å map from|Fobs|, ϕbest. The model was built into a 2.37-Å electron density map using the program O (21Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). All refinement was done with maximum likelihood amplitude and phase probability targets using CNS (22Brünger 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 (16946) Google Scholar) and the data at λ3 (Table II). Atomic occupancies of protein residues in dual positions were refined once. The final refined model consists of two IGPD monomers, A and B, related by an approximate 2-fold axis (179.5° rotation) parallel to the cubic unit cell axis. Monomers A and B reside in different IGPD trimers and give rise to the native Patterson peak. In each monomer, one short internal peptide and 14-15 C-terminal residues are disordered and missing from the model. Met-1 was not present in the crystallized protein (data not shown). Three out of four cysteine side chains were modified with mercury. No potential Mn2+ sites were identified in electron density maps. Arg-97 and Asp-106 in each monomer lie in disallowed regions of the Ramachandran plot; however, their backbone conformations are well supported by electron density. The conformation of Arg-97 (ϕ = +70, ψ = -53) is stabilized by backbone hydrogen bonds of both the NH and C=O groups, by a bidentate salt bridge to Glu-115 and by stacking with the Tyr-98 side chain. The conformation of Asp-106 (ϕ = +47, ψ = -116) is stabilized by its position in a type II β-turn (residues 105-108) and by a hydrogen bond of the backbone C=O and side chain of His-157 in an adjacent monomer. Arg-97, Asp-106, Glu-115, and His-157 are invariant among IGPD sequences, suggesting that these residues as well as their conformations may be important for IGPD function. The model is available in the Protein Data Bank with accession code 1RHY.Table IICrystallographic refinementHg-IGPDFree enzymeData range (Å)20.0-2.330.0-2.6Data cutoffI/σI > 0I/σI > 0RworkaRfactor=∑h|Fobs|-|Fcalc|/∑h|Fobs|.0.1890.202RfreeaRfactor=∑h|Fobs|-|Fcalc|/∑h|Fobs|.0.2280.251Residues, monomer A2-66, 73-1872-65, 73-188Residues, monomer B2-65, 73-1882-65, 73-188Mercury6Sulfate ions54Acetate ions71Glycerol70Water247148r.m.s.d.br.m.s.d., root mean square deviation. from target valuesBond lengths0.010 Å0.009 ÅBond angles1.7°1.59°ΔB between bonded atoms5.1 Å23.0 Å2Average B values (Å2)Protein43.246.9Solvent71.963.1r.m.s.d. between monomers0.41 Å, 167 Cα0.58 Å, 172 Cαa Rfactor=∑h|Fobs|-|Fcalc|/∑h|Fobs|.b r.m.s.d., root mean square deviation. Open table in a new tab Crystals of the free enzyme and those grown in presence of inhibitors HTPP or IRL-1803 were isomorphous with mercury-IGPD crystals. For each of these structures, the starting model for refinement was based on the mercury-IGPD structure. The three structures are virtually identical. The most reliable model was obtained from crystals grown without inhibitor (Table II). No density was observed for Mn2+, HTPP, or IRL-1803 in these structures. The absence of bound Mn2+ was confirmed by the examination of anomalous difference Fourier maps. The discussion is limited to the higher resolution mercury-IGPD structure because there are no significant differences between the mercury-IGPD and free-enzyme structures (root mean square deviation is 0.38 Å for 180 Cα atoms). Modeling of an IGPD 24-Mer—Models of IGPD 24-mers with octahedral symmetry were constructed from the crystallographic trimers. Using the trimer as a rigid body, a two-dimensional search was done through 120° rotation in 10° steps around the trimer 3-fold axis and through 140-Å translation in 5-Å steps along the trimer 3-fold. This search covers all of the rotation space, and a translation in which the ends of the range have no intertrimer contacts. At each grid position, a 24-mer was constructed from the trimer by application of 4- and 2-fold symmetry. Solutions were scored by tabulating the number of “bad” (d < 2.5 Å) and “good”(2.5 Å < d < 3.6 Å) interatomic contacts between trimers and the ratio of good to bad contacts. Full atomic coordinates were used for the search, which was done with trimers constructed from both the crystallographic A and B monomers. Most of the search space yielded impossible solutions with interpenetrating trimers. Fine searches (1° rotation step, 1-Å translation step) were done around six solutions with good:bad ratios of 3.5-16. Solutions were evaluated by visual inspection. Structure Determination—F. neoformans IGPD produced in E. coli had a heterogeneous metal content and yielded poorly diffracting crystals. To improve crystal quality and to label the protein with a single anomalous scatterer, IGPD was demetallated to produce the catalytically inactive 70-kDa species and then reconstituted with each of several metals. Many of the reconstituted proteins crystallized, but only crystals of IGPD reconstituted with Mn2+ were suitable for high resolution analysis. As Mn2+ does not have an x-ray absorption edge convenient for multiwavelength anomalous diffraction, crystals were treated with mercury, and the structure was determined by mercury multiwavelength anomalous diffraction. The crystallographic model agrees well with the diffraction data and with stereochemical criteria (Table II). The estimated coordinate error is 0.29 Å (23Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2035) Google Scholar). The final 2.3-Å model includes one monomer in each of two trimers. Monomers A and B are nearly identical, except for two loops comprising 12 residues (11Chiariotti L. Nappo A.G. Carlomagno M.S. Bruni C.B. Mol. Gen. Genet. 1986; 202: 42-47Crossref PubMed Scopus (35) Google Scholar, 12Houston L.L. J. Biol. Chem. 1973; 248: 4144-4149Abstract Full Text PDF PubMed Google Scholar, 13Parker A. Medicinal Chemistry and Molecular Pharmacognosy. Purdue University, West Lafayette, IN1995: 218Google Scholar, 14Tada S. Hatano M. Nakayama Y. Volrath S. Guyer D. Ward E. Ohta D. Plant Physiol. 1995; 109: 153-159Crossref PubMed Scopus (30) Google Scholar, 15Glaser R.D. Houston L.L. Biochemistry. 1974; 13: 5145-5152Crossref PubMed Scopus (16) Google Scholar and 25Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3555) Google Scholar, 26Murzin A.G. Struct. Biol. 1995; 2: 25-26Crossref PubMed Scopus (34) Google Scholar, 27Zhou T. Daugherty M. Grishin N.V. Osterman A.L. Zhang H. Structure (Lond.). 2000; 8: 1247-1257Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 28Symmons M.F. Jones G.H. Luisi B.F. Structure (Lond.). 2000; 8: 1215-1226Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 29Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3022) Google Scholar, 30Schuck P. Perugini M.S. Gonzales N.R. Howlett G.J. Schubert D. Biophys. J. 2002; 82: 1096-1111Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 31Lebowitz J. Lewis M.S. Schuck P. Protein Sci. 2002; 11: 2067-2079Crossref PubMed Scopus (614) Google Scholar). The variable loop conformations are not influenced by crystal lattice contacts. The remaining 167 Cα atoms superimpose with a root mean square deviation of 0.4 Å. Likewise, the trimers of monomers A and B are nearly identical with a root mean square deviation of 0.4 Å for 501 Cα atoms. IGPD Fold: Gene Duplication and Unique Arrangement of a Rare Structural Motif—The IGPD polypeptide forms a single domain (Fig. 1) consisting of a bundle of four α-helices (α1-α4), sandwiched between four-stranded, mixed β-sheets (β1-β2-β4-β3 and β5-β6-β8-β7). The fold possesses an internal repeat, in which the first β-sheet and two α-helices (β1-β2-β3-α1-β4-α2, residues 2-93) have an identical topology to the second β-sheet and two α-helices (β5-β6-β7-α3-β8-α4, residues 94-187). The half-domain motif includes a rare left-handed βαβ crossover (β3-α1-β4 and β7-α3-β8). The half-domains are related by pseudo-dyad symmetry (157° rotation) such that 77 pairs of Cα atoms superimpose with a root mean square deviation of 2.1 Å (Fig. 2).Fig. 2Internal repeat in IGPD. A, stereo view of the superposition of Cα traces of the N and C-terminal halves of the IGPD monomer. The N-terminal half is traced in yellow, and the C-terminal half is in gray. All atoms are shown for conserved side chains in the C-terminal (D/N)XHHXXE motif, colored by atomic type (gray, carbon; red, oxygen; blue, nitrogen). Residues at the N- and C terminus of each half-fold are indicated. This and other superpositions were done using the program O (21Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). B, structure-based sequence alignment of the two halves of IGPD. Evidence for gene duplication and fusion is found in conservation in a multiple-sequence alignment (39Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55386) Google Scholar) of IGPD from 56 organisms, indicated as N-terminal (above) and C-terminal (below) consensus sequences. Secondary structure elements corresponding to the F. neoformans IGPD structure are indicated above and below the sequence alignment. α-Helices are represented by cylinders, β-strands by arrows, loops by black solid lines, and disordered regions by dashed lines. The degree of conservation is indicated by the background color, with invariant residues in red (white characters), 95% invariant in pink, and sites of conservative substitution in yellow. The (D/N)XHHXXE motif is outlined. This figure was made using ALSCRIPT (40Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1110) Google Scholar). aa, amino acids.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The structural repeat matches an internal sequence repeat exhibited by IGPDs from all biological sources. The sequences of residues 1-93 and 94-202 of F. neoformans IGPD are 19% identical (Fig. 2). The most conserved feature of the internal repeat is the (D/N)XHHXXE motif in loops β3-α1 (residues 69-75) and β8-α4 (residues 165-171). The repetition of sequence and structural elements suggests a gene duplication in the evolution of IGPD. Gene duplication is a recurring theme in enzymes of histidine biosynthesis. The enzymes that catalyze the two steps preceding the dehydration reaction of IGPD, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase and imidazole glycerol-phosphate synthase, also have internal repeats and are thought to have evolved by similar gene duplication and fusion events (24Lang D. Thoma R. Henn-Sax M. Sterner R. Wilmanns M. Science. 2000; 289: 1546-1550Crossref PubMed Scopus (260) Google Scholar). These enzymes are homologs but are unrelated to IGPD. The structure data base contains no proteins with folds like the IGPD fold, based on a topology search (25Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3555) Google Scholar) of the Protein Data Ba" @default.
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• W2052283610 title "Crystal Structure of Imidazole Glycerol-phosphate Dehydratase" @default.
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• W2052283610 cites W1529281090 @default.
• W2052283610 cites W1539796472 @default.
• W2052283610 cites W1570928892 @default.
• W2052283610 cites W1810161475 @default.
• W2052283610 cites W1943524665 @default.
• W2052283610 cites W1965277349 @default.
• W2052283610 cites W1969490782 @default.
• W2052283610 cites W1973360750 @default.
• W2052283610 cites W1975317507 @default.
• W2052283610 cites W1978561997 @default.
• W2052283610 cites W1986969007 @default.
• W2052283610 cites W1993016259 @default.
• W2052283610 cites W1993404146 @default.
• W2052283610 cites W1995017064 @default.
• W2052283610 cites W1995711572 @default.
• W2052283610 cites W1999746361 @default.
• W2052283610 cites W2000046980 @default.
• W2052283610 cites W2008476014 @default.
• W2052283610 cites W2009259291 @default.
• W2052283610 cites W2010693606 @default.
• W2052283610 cites W2013083986 @default.
• W2052283610 cites W2015474832 @default.
• W2052283610 cites W2022058405 @default.
• W2052283610 cites W2023200543 @default.
• W2052283610 cites W2028231353 @default.
• W2052283610 cites W2052444161 @default.
• W2052283610 cites W2057393927 @default.
• W2052283610 cites W2064715719 @default.
• W2052283610 cites W2071124827 @default.
• W2052283610 cites W2076300945 @default.
• W2052283610 cites W2080528351 @default.
• W2052283610 cites W2091969321 @default.
• W2052283610 cites W2106882534 @default.
• W2052283610 cites W2114586294 @default.
• W2052283610 cites W2117164735 @default.
• W2052283610 cites W2119966574 @default.
• W2052283610 cites W21523657 @default.
• W2052283610 cites W2163449711 @default.
• W2052283610 cites W2195878577 @default.
• W2052283610 cites W2316557352 @default.
• W2052283610 cites W2951272776 @default.
• W2052283610 cites W6261644 @default.
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