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• W2009254923 abstract "The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis. The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis. The serine (Ser)-histidine (His)-aspartate (Asp) triad is a well known catalytic motif that is utilized in catalysis of a diverse array of chemical transformations by a large number of enzymes. One group of these triad-dependent enzymes includes numerous hydrolytic enzymes in the trypsin and subtilisin families of serine peptidases that cleave C–N bonds in peptide substrates (1Page M.J. Di Cera E. Serine peptidases: classification, structure and function.Cell. Mol. Life Sci. 2008; 65: 1220-1236Crossref PubMed Scopus (289) Google Scholar). Another group of enzymes utilizing this triad or its variants are members of the α/β-hydrolase fold superfamily that catalyze cleavage of C–O, C–S, C–C, or C–halogen bonds in structurally diverse metabolites or even cofactor-independent haloperoxidation and dioxygenation of electron-rich substrates (2Nardini M. Dijkstra B.W. α/β Hydrolase fold enzymes: the family keeps growing.Curr. Opin. Struct. Biol. 1999; 9: 732-737Crossref PubMed Scopus (678) Google Scholar, 3Holmquist M. α/β-Hydrolase fold enzymes: structures, functions and mechanisms.Curr. Protein Pept. Sci. 2000; 1: 209-235Crossref PubMed Scopus (482) Google Scholar). Commensurate with their functional diversity, these enzymes display vast structural diversity in size, structure fold, and three-dimensional architecture. However, they converge to form the conserved triad and the associated oxyanion hole as a stable, integrative catalytic unit from distal residues, which are arranged in one enantiomeric configuration in α/β-hydrolase fold enzymes and in another configuration in members of the trypsin and subtilisin families (3Holmquist M. α/β-Hydrolase fold enzymes: structures, functions and mechanisms.Curr. Protein Pept. Sci. 2000; 1: 209-235Crossref PubMed Scopus (482) Google Scholar). Besides this structural conservation, a majority of these enzymes use the triad to catalyze the diverse chemical transformations via a similar nucleophilic mechanism, which is characterized by formation of a covalent enzyme adduct involving nucleophilic addition of the triad serine to the substrate (3Holmquist M. α/β-Hydrolase fold enzymes: structures, functions and mechanisms.Curr. Protein Pept. Sci. 2000; 1: 209-235Crossref PubMed Scopus (482) Google Scholar, 4Hedstrom L. Serine protease mechanism and specificity.Chem. Rev. 2002; 102: 4501-4524Crossref PubMed Scopus (1336) Google Scholar). Besides the traditional nucleophilic mechanism of catalysis, the Ser-His-Asp triad has also been proposed to play the role of a simple general base in the catalysis of some α/β-hydrolases. In the catalysis of cyanohydrin lyases, the triad is postulated to deprotonate hydrogen cyanide for addition to aldehydes and ketones (5Wajant H. Pfizenmaier K. Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis.J. Biol. Chem. 1996; 271: 25830-25834Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 6Zuegg J. Gruber K. Gugganig M. Wagner U.G. Kratky C. Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.Protein Sci. 1999; 8: 1990-2000Crossref PubMed Scopus (69) Google Scholar) despite the fact that this triad is able to form a covalent complex with trichloroacetaldehyde through nucleophilic addition (6Zuegg J. Gruber K. Gugganig M. Wagner U.G. Kratky C. Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.Protein Sci. 1999; 8: 1990-2000Crossref PubMed Scopus (69) Google Scholar). A similar general base role was also proposed for the triad of C–C bond hydrolases (7Fleming S.M. Robertson T.A. Langley G.J. Bugg T.D. Catalytic mechanism of a C–C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme.Biochemistry. 2000; 39: 1522-1531Crossref PubMed Scopus (55) Google Scholar, 8Speare D.M. Fleming S.M. Beckett M.N. Li J.J. Bugg T.D. Synthetic 6-aryl-2-hydroxy-6-keto-hexa-2,4-dienoic acid substrates for C–C hydrolase BphD: investigation of a general base catalytic mechanism.Org. Biomol. Chem. 2004; 2: 2942-2950Crossref PubMed Google Scholar, 9Li J.-J. Li C. Blindauer C.A. Bugg T.D. Evidence for a gem-diol reaction intermediate in bacterial C–C hydrolase enzymes BphD and MhpC from 13C NMR spectroscopy.Biochemistry. 2006; 45: 12461-12469Crossref PubMed Scopus (27) Google Scholar), which are involved in the degradation pathways of aromatic compounds. However, recent investigations have shown that the C–C bond hydrolase BphD adopts the traditional covalent, nucleophilic mechanism of catalysis because of the finding of an acyl-enzyme intermediate (10Ruzzini A.C. Ghosh S. Horsman G.P. Foster L.J. Bolin J.T. Eltis L.D. Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad.J. Am. Chem. Soc. 2012; 134: 4615-4624Crossref PubMed Scopus (30) Google Scholar, 11Ruzzini A.C. Horsman G.P. Eltis L.D. The catalytic serine of MCP hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage.Biochemistry. 2012; 51: 5831-5840Crossref PubMed Scopus (15) Google Scholar). Noticeably, the triad serine in these enzymes has been demonstrated to exhibit nucleophilic reactivity (5Wajant H. Pfizenmaier K. Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis.J. Biol. Chem. 1996; 271: 25830-25834Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Li J.-J. Bugg T.D.H. Investigation of a general base mechanism for ester hydrolysis in C–C hydrolase enzymes of the α/β-hydrolase superfamily: a novel mechanism for the serine catalytic triad.Org. Biomol. Chem. 2007; 5: 507-513Crossref PubMed Google Scholar). In addition, a nucleophilic mechanism of catalysis is able to explain the reactions catalyzed by these enzymes equally well as the general base mechanism of catalysis (10Ruzzini A.C. Ghosh S. Horsman G.P. Foster L.J. Bolin J.T. Eltis L.D. Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad.J. Am. Chem. Soc. 2012; 134: 4615-4624Crossref PubMed Scopus (30) Google Scholar, 13Wagner U.G. Hasslacher M. Griengl H. Schwab H. Kratky C. Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis.Structure. 1996; 4: 811-822Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Recently, the vitamin K biosynthetic enzyme (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) 2The abbreviations used are:SHCHC(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylateMenH(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthaseSEPHCHC(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylater.m.s.d.root mean square difference. synthase (MenH) has been shown to use its Ser-His-Asp triad as a general base. This enzyme is also a typical α/β-hydrolase fold enzyme and is responsible for 2,5-elimination of pyruvate from (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) to form SHCHC (14Jiang M. Chen X. Guo Z.-F. Cao Y. Chen M. Guo Z. Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli.Biochemistry. 2008; 47: 3426-3434Crossref PubMed Scopus (63) Google Scholar), which is a chemical conversion difficult to adopt a nucleophilic mechanism of catalysis commonly utilized by serine proteases and other α/β-fold hydrolases. The MenH catalytic triad most likely catalyzes simple abstraction of a proton from the α-carbon of the succinyl carbonyl group of the substrate to form an enolate intermediate that subsequently undergoes elimination of the enolpyruvyl group in an E1cb mechanism (Fig. 1) (15Jiang M. Chen X. Wu X.-H. Chen M. Wu Y.D. Guo Z. Catalytic mechanism of SHCHC synthase in the menaquinone biosynthesis of Escherichia coli: identification and mutational analysis of the active site residues.Biochemistry. 2009; 48: 6921-6931Crossref PubMed Scopus (29) Google Scholar). This distinctive MenH catalytic mechanism demonstrates that the classical Ser-His-Asp triad is indeed capable of new catalytic chemistry. (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate root mean square difference. Despite its unique mechanism of catalysis, the MenH triad is not found to be structurally different from that of other triad-utilizing enzymes (Refs. 14Jiang M. Chen X. Guo Z.-F. Cao Y. Chen M. Guo Z. Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli.Biochemistry. 2008; 47: 3426-3434Crossref PubMed Scopus (63) Google Scholar, 17Dawson A. Fyfe P.K. Gillet F. Hunter W.N. Exploiting the high-resolution crystal structure of Staphylococcus aureus MenH to gain insight into enzyme activity.BMC Struct. Biol. 2011; 11: 19Crossref PubMed Scopus (12) Google Scholar, and 18Johnston J.M. Jiang M. Guo Z. Baker E.N. Crystal structures of E. coli native MenH and two active site mutants.PLoS One. 2013; 8: e61325Crossref PubMed Scopus (9) Google Scholar and Protein Data Bank code 1R3D). In addition, mutational studies found that the MenH catalytic triad contributes to rate enhancement of at least 106-fold to the catalysis of the enzyme (14Jiang M. Chen X. Guo Z.-F. Cao Y. Chen M. Guo Z. Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli.Biochemistry. 2008; 47: 3426-3434Crossref PubMed Scopus (63) Google Scholar), similar to the catalytic contribution of the triads of serine proteases. At present, it is not clear why the structurally similar triad plays the role of a catalytic base in MenH catalysis but a nucleophile in catalysis of other triad-dependent enzymes. To further understand the catalytic mechanism of MenH, we determined the crystal structure of the enzyme with and without its products. We found that formation of the catalytic triad is subject to control by an open-closed conformational change. This finding not only provides new insights into the MenH catalytic mechanism but also reveals a new mechanism for control and modulation of reactivity of the catalytic triad. The expression constructs of the mutants of Escherichia coli MenH, including V152A, V152G, F153A, Y148F, Y148A, V152G/F153A, and W147A/Y148A, were generated with a QuikChange site-directed mutagenesis kit (Stratagene). All mutants were sequenced to ensure that only the expected mutations had been incorporated into the amplified DNA. The wild-type MenH and its mutants were expressed and purified according to a procedure reported previously (15Jiang M. Chen X. Wu X.-H. Chen M. Wu Y.D. Guo Z. Catalytic mechanism of SHCHC synthase in the menaquinone biosynthesis of Escherichia coli: identification and mutational analysis of the active site residues.Biochemistry. 2009; 48: 6921-6931Crossref PubMed Scopus (29) Google Scholar). Briefly, the expression plasmid for a target protein was transformed into E. coli strain BL21(DE3), and the resulting recombinant cells were grown in Luria broth (LB) containing 100 μg/ml ampicillin. For expression of 15N-labeled MenH, the cells were grown in M9 minimum medium containing 1g/liter [15N]NH4Cl and 100 μg/ml ampicillin. Overexpression of all proteins was induced using 0.2 mm isopropyl 1-thio-β-d-galactopyranoside for 16 h at 18 °C. The recombinant proteins were purified to greater than 95% purity as indicated by SDS-PAGE via Ni2+-chelating column chromatography followed by size exclusion chromatography. The purified unlabeled proteins were concentrated and stored in 25 mm Tris-HCl, pH 8.0 and 10% glycerol for crystallization or activity assays, whereas the labeled protein was stored in 50 mm sodium phosphate buffer containing 50 mm NaCl and 10% glycerol at pH 7.4. Protein concentration was estimated using a Coomassie Blue protein assay kit (Pierce). The reaction product of MenH, SHCHC, was prepared using a chemoenzymatic method described previously (14Jiang M. Chen X. Guo Z.-F. Cao Y. Chen M. Guo Z. Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli.Biochemistry. 2008; 47: 3426-3434Crossref PubMed Scopus (63) Google Scholar) from chorismic acid and 2-ketoglutarate using recombinant EntC (19Jiang M. Guo Z. Effects of macromolecular crowding on the intrinsic catalytic efficiency and structure of enterobactin-specific isochorismate synthase.J. Am. Chem. Soc. 2007; 129: 730-731Crossref PubMed Scopus (95) Google Scholar, 20Guo Z.-F. Jiang M. Zheng S. Guo Z. Suppression of linear side products by macromolecular crowding in nonribosomal enterobactin biosynthesis.Org. Lett. 2008; 10: 649-652Crossref PubMed Scopus (11) Google Scholar), MenD (21Jiang M. Cao Y. Guo Z.-F. Chen M. Chen X. Guo Z. Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) as a novel intermediate and re-evaluation of MenD activity.Biochemistry. 2007; 46: 10979-10989Crossref PubMed Scopus (84) Google Scholar), MenC (21Jiang M. Cao Y. Guo Z.-F. Chen M. Chen X. Guo Z. Menaquinone biosynthesis in Escherichia coli: identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) as a novel intermediate and re-evaluation of MenD activity.Biochemistry. 2007; 46: 10979-10989Crossref PubMed Scopus (84) Google Scholar), and MenH (14Jiang M. Chen X. Guo Z.-F. Cao Y. Chen M. Guo Z. Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli.Biochemistry. 2008; 47: 3426-3434Crossref PubMed Scopus (63) Google Scholar). Typically, a mixture of 12.5 mm chorismate, 50 mm 2-ketoglutarate, 75 μm thiamine diphosphate, and 7.5 mm MgSO4 was incubated with 30 μm EntC, 30 μm MenD, and 15 μm MenH in 200 mm sodium phosphate buffer at 32 °C for 3 h. The resulting SHCHC solution was then purified by HPLC using an isocratic elution with aqueous solution containing 1% formic acid. The enzyme substrate SEPHCHC was synthesized similarly in the absence of MenH (22Jiang M. Chen M. Cao Y. Yang Y. Sze K.H. Chen X. Guo Z. Determination of the stereochemistry of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid, a key intermediate in menaquinone biosynthesis.Org. Lett. 2007; 9: 4765-4767Crossref PubMed Scopus (27) Google Scholar). Chorismic acid was extracted from the metabolites of an engineered bacterial strain (23Grisostomi G. Kast P. Pulido R. Huynh J. Hilvert D. Efficient in vivo synthesis and rapid purification of chorismic acid using an engineered Escherichia coli strain.Bioorg. Chem. 1997; 25: 297-305Crossref Scopus (42) Google Scholar). The assay for the SHCHC synthase activity of MenH has been described previously (14Jiang M. Chen X. Guo Z.-F. Cao Y. Chen M. Guo Z. Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli.Biochemistry. 2008; 47: 3426-3434Crossref PubMed Scopus (63) Google Scholar). Briefly, the reactions were carried out in 50 mm sodium phosphate buffer at pH 7.0 containing the SEPHCHC substrate at varied concentrations. MenH and its mutants were then added to the mixture to initiate the reaction. Production of SHCHC was monitored in real time by the increase in absorbance at 290 nm. One-dimension NMR spectra were acquired on a Varian UNITY Inova 500-MHz NMR spectrometer equipped with an actively z-gradient-shielded triple resonance probe where the water signal was suppressed using the WATERGATE sequence. For each spectrum, 1024 scans were acquired consisting of 128,000 data points with a spectral width of 35 ppm at 4 °C. The concentration of MenH or the H232A mutant was fixed at 0.33 mm, and SHCHC was titrated into the MenH solution to a concentration of 0.165, 0.33, and 0.66 mm. As a control, one-dimension proton NMR was collected for 0.36 mm SHCHC alone. To determine which of the protons at δH = 19.10 and 14.16 ppm is hydrogen-bonded to the imidazole side chain of His232, a mixture of 0.40 mm 15N-labeled MenH and 0.80 mm SHCHC was used for analysis by heteronuclear single quantum coherence spectroscopy. However, no cross-peaks were found probably because of the weak 15N signals. The dissociation constant (KD) for binding of the SHCHC ligand by MenH or its H232A mutant was determined by the ligand-dependent quenching of tryptophan fluorescence with a PerkinElmer Life Sciences Model LS-55 luminescence spectrometer. Specifically, the SHCHC ligand was titrated into the solution of MenH or the H232A mutant at 2.0 μm in the 25 mm Tris-HCl buffer, pH 8.0. The tryptophan fluorophore was excited at 280 nm, and its emission (F) at 340 nm was recorded. The dissociation constant was determined by the least square fitting of the fluorescence-[SHCHC] curves according to the equation F/F0 = KD/(KD + [SHCHC]) where F0 is the tryptophan fluorescence of the protein in the absence of any ligands. Rapid kinetic measurements of either UV-visible absorption or fluorescence emission were performed on an Applied Photophysics SX.18MV-R stopped-flow reaction analyzer mounted with an Applied Photophysics spectral kinetic monochromator. In pre-steady state kinetic measurements, 50-μl aliquots of MenH at 2.0 μm were mixed with an equal volume of SEPHCHC solution at concentrations varying from 5.0 to 240 μm. The release of the SHCHC product was monitored at 290 nm for a total of 200 ms with a 1-ms dead time. In the kinetic measurement of SHCHC binding, 50-μl aliquots of MenH at 1.0 μm were mixed with an equal volume of SHCHC at concentrations varying from 0 to 360 μm. Quenching of the tryptophan fluorescence emission at 340 nm was determined over a period of 200 ms as a change of fluorescence voltage (photomultiplier tube voltage) using a 305-nm high pass filter with a monochromatic excitation wavelength of 280 nm. The buffer used in the stopped-flow experiments was 0.20 m phosphate buffer at pH 7.0. The slit width of the monochromator source was set to 8 nm in all experiments. Initial screening of crystallization conditions for ligand-free MenH and MenH in complex with SHCHC was performed using hanging drop vapor diffusion with a range of screen kits (Hampton Research) at 293 K. The concentrations of MenH and SHCHC were 10 mg/ml and 2 mm, respectively. The protein solution was mixed with the reservoir solution in a 1:1 ratio in these screening trials. Small ellipsoid and rhombohedral crystals were obtained within 1 week under the same conditions for both the complex and the ligand-free protein. After several rounds of optimization, the longest dimension of the crystals reached about 100 μm. However, neither the complex nor the ligand-free protein crystals diffracted to a high resolution on an in-house x-ray diffractometer. After optimization via additive screens using the Crystal Screen kit and Crystal Screen II kit (Hampton Research), cuboid-shaped crystals of ligand-free MenH were observed in solution containing 0.2 m Li2SO4, 0.1 m Tris buffer, pH 9.0, 30% (v/v) glycerol, and 17% PEG 3350 with a dimension of 300 × 300 × 200 μm. Large, rod-shaped crystals (500 μm for the longest dimension) of the MenH complex were obtained when the initial screens were rechecked 2 months later in solution containing 1.6 m sodium/potassium phosphate, pH 6.9. The ternary complex was crystallized under the same conditions as for the binary complex with the addition of 2 mm pyruvate. The crystals were mounted and soaked in cryoprotectant containing 20% glycerol in the mother liquor and then flash frozen in liquid N2. X-ray diffraction data were collected for the crystals of ligand-free MenH and the complexes at beamline BL17U at Shanghai Synchrotron Radiation Facility with an ADSC Quantum 315R charge-coupled device detector. Diffraction images were indexed, integrated, and scaled using HKL2000 (24Otwinowski Z. Minor W. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38527) Google Scholar). The ligand-free MenH structure was solved by molecular replacement with Phaser (25McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14444) Google Scholar) in the CCP4 suite (26Collaborative Computational Project, Number 4 The CCP4 suite: programs for protein crystallography.Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) using the structure of Vibrio cholerae MenH (Protein Data Bank code 1R3D) as the search model. The initial electron density map indicated that one MenH polypeptide chain was correctly located in the asymmetric unit in space group P3121. The model was then extended via automatic model building using the program ARP/wARP (27Langer G. Cohen S.X. Lamzin V.S. Perrakis A. Automated macromolecular model building for x-ray crystallography using ARP/wARP version 7.Nat. Protoc. 2008; 3: 1171-1179Crossref PubMed Scopus (1319) Google Scholar) and then further built manually using Coot (28Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17085) Google Scholar) followed by refinement using REFMAC (29Vagin A.A. Steiner R.A. Lebedev A.A. Potterton L. McNicholas S. Long F. Murshudov G.N. REFMAC5 dictionary: organisation of prior chemical knowledge and guidelines for its use.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2184-2195Crossref PubMed Scopus (1064) Google Scholar) and PHENIX (30Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. Richardson J.S. Terwilliger T.C. Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar). The overall quality of the structural model was assessed by PROCHECK (31Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. PROCHECK: a program to check the stereochemical quality of protein structures.J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and MolProbity (32Chen V.B. Arendall 3rd, W.B. Headd J.J. Keedy D.A. Immormino R.M. Kapral G.J. Murray L.W. Richardson J.S. Richardson D.C. MolProbity: all-atom structure validation for macromolecular crystallography.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 12-21Crossref PubMed Scopus (9818) Google Scholar). Data collection and refinement statistics are summarized in Table 1.TABLE 1Data collection and refinement statisticsMenHMenH-SHCHCMenH-SHCHC-pyruvatePDB code4MXD4MYD4MYSData collectionSpace groupP3121P31P31Unit cell dimensionsa, b, c (Å)72.2, 72.2, 112.6121.9, 121.9, 46.9122.0, 122.0, 46.9α, β, γ (°)90, 90, 12090, 90, 12090, 90, 120Redundancy10.8 (10.9)5.3 (4.9)5.0 (4.6)Completeness (%)99.9 (100)99.7 (99.7)99.5 (97.4)Reflections (unique)656,511 (60,673)855,411 (161,920)729,480 (145,860)I/σI16.0 (5.3)9.0 (2.3)10.7 (2.9)Rmerge0.091 (0.59)0.083 (0.69)0.090 (0.41)RefinementResolution range (Å)50–1.4546.8–1.3737.2–1.42No. of atoms2,4588,5798,596Macromolecules2,0697,7487,724Water351797826Ligands/ions383446Average B factor (Å2)22.028.024.9Macromolecules19.523.221.4Water35.141.937.4LigandN/A12.2aValue for the SHCHC ligand.9.9/20.9bValues for SHCHC/pyruvate.Rwork/Rfree (%)13.2/15.012.8/16.617.4/19.5r.m.s.d. for ideal valueBond length (Å)0.0080.010.006Bond angle (°)1.301.201.08Ramachandran plotFavored/allowed/outliers (%)98.9/1.1/099.0/1.0/099.0/1/0a Value for the SHCHC ligand.b Values for SHCHC/pyruvate. Open table in a new tab Based on the diffraction patterns, the MenH-SHCHC complex crystal is in the P3121 space group. The crystal structure solved by molecular replacement with Phaser using the ligand-free MenH structure as the search model contained a high solvent content of 66.1% with one-third of the crystal volume occupied by non-interpretable electron densities. Although the unit cell contains MenH molecules with very well defined electron densities, this structure did not appear to be a correct solution. The diffraction data set was then reindexed to the P31 space group for structural determination by molecular replacement using Phaser and BALBES (33Long F. Vagin A.A. Young P. Murshudov G.N. BALBES: a molecular replacement pipeline.Acta Crystallogr. D Biol. Crystallogr. 2008; 64: 125-132Crossref PubMed Scopus (631) Google Scholar). The resulting structure contained two well defined MenH molecules in the asymmetric unit, which also contained uninterpreted electron densities in one-third of its volume. A third MenH molecule was successfully generated in the asymmetric unit after model building by OASIS (34Zhang T. Gu Y.-X. Zheng C.-D. Fan H.-F. OASIS4.0—a new version of the program OASIS for phasing protein diffraction data.Chin. Phys. B. 2010; 19 (086101)Google Scholar) albeit with poor and disconnected electron densities. By applying the merohedral twin law (h, −h−k, −l), the model was eventually refined by Phenix.refine to a high resolution with Rwork/Rfree = 0.13/0.15. However, the actual Rwork/Rfree was found to be 0.23/0.25 in the validation process, indicative of the absence of twinning in the crystal. Examination of the reflection images and the self-rotation function found that the crystal likely had an order-disorder structure (35Hare S. Cherepanov P. Wang J. Application of general formulas for the correction of a lattice-translocation defect in crystals of a lentiviral integrase in complex with LEDGF.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 966-973Crossref PubMed Google Scholar, 36Pletnev S. Morozova K.S. Verkhusha V.V. Dauter Z. Rotational order-disorder structure of fluorescent protein FP480.Acta Crystallogr. D" @default.
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• W2009254923 title "Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad" @default.
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