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• W2099381109 abstract "Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the α/β hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen. Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the α/β hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen. Epoxide hydrolases (EC 3.3.2.3) are a group of functionally related enzymes that catalyze the cofactor-independent hydrolysis of epoxides to their corresponding diols by the addition of a water molecule. Epoxides are very reactive electrophilic compounds frequently found as intermediates in the catabolic pathway of various xenobiotics. For instance they are the carcinogens formed by bioactivation reactions catalyzed by cytochrome P450. Therefore, conversion of epoxides to less toxic, water-soluble compounds is an essential detoxification step in living cells. Consequently, epoxide hydrolases have been found in a wide variety of organisms, including mammals, invertebrates, plants, and bacteria (1Archelas A. Furstoss R. Trends Biotechnol. 1998; 16: 108-116Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Until now most research has been focused on mammalian epoxide hydrolases (2Lacourciere G.M. Armstrong R.N. Chem. Res. Toxicol. 1994; 7: 121-124Crossref PubMed Scopus (51) Google Scholar, 3Arand M. Grant D.F. Beetham J.K. Friedberg T. Oesch F. Hammock B.D. FEBS Lett. 1994; 338: 251-256Crossref PubMed Scopus (133) Google Scholar), which, together with glutathioneS-transferases, are the most important enzymes to convert toxic epoxides to more polar and easily excretable compounds (4Herandez O. Bend J.R. Metabolic Basis of Detoxication. Academic Press, Inc., New York1982: 207-228Crossref Google Scholar). However, much progress has recently also been made in the characterization of bacterial epoxide hydrolases (5Jacobs M.H.J. van den Wijngaard A.J. Pentenga M. Janssen D.B. Eur. J. Biochem. 1991; 202: 1217-1222Crossref PubMed Scopus (70) Google Scholar, 6Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 7Misawa E. Chion C.K.C.C.K. Archer I.V. Woodland M.P. Zhou N.Y. Carter S.F. Widdowson D.A. Leak D.J. Eur. J. Biochem. 1998; 253: 173-183Crossref PubMed Scopus (46) Google Scholar). These enzymes show a significant sequence homology with those of mammalian origin. They can be easily obtained in large amounts, and they exhibit enantioselectivity with various industrially important epoxides, which makes them promising biocatalysts for the large scale preparation of enantiopure epoxides and/or their corresponding vicinal diols (8Lutje Spelberg J.H. Rink R. Kellogg R.M. Janssen D.B. Tetrahedron: Asymmetry. 1998; 9: 459-466Crossref Scopus (90) Google Scholar). In particular, extensive studies have been performed on the epoxide hydrolase from Agrobacterium radiobacter AD1, a Gram-negative bacterium that is able to use the environmental pollutant epichlorohydrin as its sole carbon and energy source (5Jacobs M.H.J. van den Wijngaard A.J. Pentenga M. Janssen D.B. Eur. J. Biochem. 1991; 202: 1217-1222Crossref PubMed Scopus (70) Google Scholar, 6Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 8Lutje Spelberg J.H. Rink R. Kellogg R.M. Janssen D.B. Tetrahedron: Asymmetry. 1998; 9: 459-466Crossref Scopus (90) Google Scholar). This epoxide hydrolase is a soluble monomeric globular protein of 35 kDa with a broad substrate range. Epichlorohydrin and epibromohydrin are its best substrates, and the optimum pH range for catalysis is 8.4–9.0. Sequence and secondary structure analysis suggested that this enzyme belongs to the α/β hydrolase-fold family of enzymes (9Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1821) Google Scholar). Site-specific mutations indicated Asp107, His275, and Asp246 as the catalytic triad residues. The proposed catalytic mechanism involves two steps analogous to haloalkane dehalogenase (10Verschueren K.H.G. Seljée F. Rozeboom H.J. Kalk K.H. Dijkstra B.W. Nature. 1993; 363: 693-698Crossref PubMed Scopus (416) Google Scholar). In the first reaction step, an ester bond is formed between enzyme and substrate by attack of the nucleophilic Asp107 on the primary carbon atom of the substrate; in the second step, this ester bond is hydrolyzed by a water molecule activated by the His275/Asp246 pair. The reaction proceeds via two different transition states, one during the binding and opening of the epoxide ring and the second during the hydrolysis of the ester intermediate. However, several important questions remained unanswered. Until now it has not been possible to identify the residue responsible for the binding and protonation of the epoxide oxygen, nor was the location known of the oxyanion hole that stabilizes the Asp107 oxyanion during the hydrolysis of the ester intermediate. Structural information may also resolve why an Asp246 → Ala mutant still retains some residual activity (6Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Here we report the 2.1-Å resolution x-ray structure of the epoxide hydrolase from A. radiobacter AD1 (Ephy). 1The abbreviations used are: Ephy, epoxide hydrolase from A. radiobacter AD1; DhlA, haloalkane dehalogenase from X. autotrophicus GJ10; Pgl, triacylglycerol lipase from P. glumae ; Hpl, human pancreatic lipase; NCS, noncrystallographic symmetry; r.m.s., root mean square. 1The abbreviations used are: Ephy, epoxide hydrolase from A. radiobacter AD1; DhlA, haloalkane dehalogenase from X. autotrophicus GJ10; Pgl, triacylglycerol lipase from P. glumae ; Hpl, human pancreatic lipase; NCS, noncrystallographic symmetry; r.m.s., root mean square. It is the first epoxide hydrolase for which the structure has been solved. The result of this work can provide a general better understanding about the structural basis of the reaction mechanism for this class of important ubiquitous enzymes. The epoxide hydrolase from A. radiobacter AD1 was cloned, overexpressed, and purified as described previously (6Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The stock protein solution, containing 5 mm potassium phosphate, 1 mm EDTA, 1 mm β-mercaptoethanol, 0.02% sodium azide, and 10% glycerol (pH 6.8) was concentrated and extensively washed using 10 mm potassium phosphate (pH 7.0) in a Centricon-10 ultracentrifugation concentrator with a 10-kDa cut-off (Amicon) to a final protein concentration of 5.5 mg/ml. The extensive washing procedure is essential to remove the glycerol before crystallization. The glycerol induces high polydispersity in the protein sample, as was determined by dynamic light scattering analysis on a DynaPro 801 instrument (Protein Solutions, Charlottesville, VA). Removing the glycerol resulted in a solution containing particles with a 2.7-nm diameter (apparent molecular mass of 33 kDa) and a polydispersity of 15%. Crystallization experiments with the protein in the presence of glycerol gave only very thin needles as a best result. An initial promising crystallization condition was obtained from a Sparse Matrix screening (11Jancarik J. Kim S.H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2076) Google Scholar). Refinement of this Sparse Matrix condition resulted in the following crystallization protocol: hanging drops (4 μl of protein solution and 4 μl of precipitant) were equilibrated against a 1-ml reservoir containing 1.6 to 1.8 mKH2PO4/K2HPO4 (pH 7.0) at room temperature. After 2 weeks, the experiments were allowed to slowly evaporate to a phosphate concentration of about 2.0m. The slow increase of the phosphate concentration in the drop results in the appearance of crystals with typical sizes of 0.3 × 0.2 × 0.1 mm3. They are highly x-ray-sensitive, and therefore, all data collections were performed at cryotemperature (100 K), using 30% glycerol added to the stabilizing mother liquor (1.8 mKH2PO4/K2HPO4) as a cryoprotectant. The crystals diffract up to 2.1-Å resolution using synchrotron radiation, and they belong to space group C2 with unit cell parameters of a = 146.62 Å, b = 100.20 Å, c = 96.88 Å, β = 100.68°. This unit cell gives a V M value of 2.57 Å3/Da−1assuming 4 molecules in the asymmetric unit. The deduced solvent content of the crystals is 52%. Heavy atom derivatives were prepared by soaking the crystals in solutions obtained by dissolving the heavy atom compounds in the standard mother liquor (1.8 mKH2PO4/K2HPO4). The search resulted in only one good isomorphous derivative obtained by soaking a crystal of epoxide hydrolase for 2 days in a solution of 2.0 mm ethyl mercury phosphate, (C2H5HgO)3PO. A 2.1-Å resolution native data set and one of the two ethyl mercury phosphate derivative data sets were collected at the x-ray diffraction beamline of the ELETTRA synchrotron in Trieste (Italy), equipped with a 30-cm MAR image plate area detector (MAR Research, Hamburg, Germany) with the wavelength tuned to λ = 1.0 Å. An in-house derivative data set was collected on a Mac Science DIP-2030H area detector equipped with a dual 30-cm image plate, with graphite monochromatized CuKα radiation (λ = 1.5418 Å) from a FR591 rotating anode generator with a double mirror x-ray focusing system (model MAC-XOS) as x-ray source (Enraf Nonius, Delft, The Netherlands). All data sets were collected at 100 K, integrated, and merged using the DENZO/SCALEPACK package (12Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar) and software from the BIOMOL crystallographic package (Protein Crystallography Group, University of Groningen). Derivative data were scaled to the native data set using the program PHASES (13Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar). Data-processing statistics are given in Table I.Table IData collection and single isomorphous replacement including anomalous scattering (SIRAS) analysisData setNative (λ = 1.0 Å)HgaThe Hg derivative is ethyl mercury phosphate (C2H5HgO)3PO. (λ = 1.0 Å)HgaThe Hg derivative is ethyl mercury phosphate (C2H5HgO)3PO. (λ = 1.5418 Å)Space groupC2C2C2a =146.62 Å147.15 Å147.05 Åb =100.20 Å100.14 Å100.33 Åc =96.88 Å97.70 Å97.32 Åβ =100.68°100.94°100.51°Resolution2.1 Å3.5 Å4.0 ÅObservations222,88065,94159,453Unique reflections73,44515,78210,514Completeness (%) overall (final shell)91.5 (82.0)93.9 (91.7)88.3 (91.2)R merge (final shell)bR merge = ∑h∑i‖I hi − 〈I h〉‖/∑h∑i I hi.0.06 (0.36)0.13 (0.23)0.12 (0.25)PhasingHeavy atom sites2222Phasing power (iso/ano)cPhasing power is r.m.s. (‖F H‖/E), where ‖F H‖ is the heavy atom structure factor amplitude, and E is the radial lack of closure (‖F PH − F P‖ − ‖F H‖). iso, isomorphous difference; ano, anomalous difference.1.80/1.872.01Figure of merit (iso/ano)dFigure of merit for a given reflection h ism = ‖F best‖/‖F h‖.0.35/0.400.42Figure of merit overall0.47R CulliseR Cullis = ∑h∥F PH‖±‖Fp‖ −F H(calc)‖/∑h‖F PH± F P∥.0.490.43a The Hg derivative is ethyl mercury phosphate (C2H5HgO)3PO.b R merge = ∑h∑i‖I hi − 〈I h〉‖/∑h∑i I hi.c Phasing power is r.m.s. (‖F H‖/E), where ‖F H‖ is the heavy atom structure factor amplitude, and E is the radial lack of closure (‖F PH − F P‖ − ‖F H‖). iso, isomorphous difference; ano, anomalous difference.d Figure of merit for a given reflection h ism = ‖F best‖/‖F h‖.e R Cullis = ∑h∥F PH‖±‖Fp‖ −F H(calc)‖/∑h‖F PH± F P∥. Open table in a new tab The structure of the epoxide hydrolase from A. radiobacter AD1 was solved by the method of single isomorphous replacement supplemented by anomalous scattering, using both the in-house and synchrotron derivative data sets. A major heavy atom site (8.5 ς) for the ethyl mercury phosphate derivative was located in a difference Patterson map (12.0–4.5-Å data). The remaining 21 heavy atom positions were determined using difference Fourier techniques. Heavy atom position search, parameter refinement including anomalous data, and phase calculations were performed with PHASES (13Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar) (Table I). The initial phases calculated at 3.7 Å yielded a figure of merit of 0.47 and were improved by solvent flattening and histogram matching techniques using the program DM (14Collaborative Computational Project Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar). The noncrystallographic symmetry (NCS) operators (three orthogonal 2-fold axes) relating the 4 molecules in the asymmetric unit were determined with the help of FINDNCS, 2G. Lu,http://gamma.mbb.ki.se/∼guoguang/findncs.html. using the 8 heavy atom sites with the highest occupancies. They were checked by comparing them with the rotation matrices calculated from a self-rotation function (16Navaza J. Saludjian P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar). An initial mask was built around one molecule in the asymmetric unit with the program MAMA (17Kleywegt G.J. Jones T.A. Acta Crystallogr. Sec. D. 1996; 52: 842-857Crossref PubMed Scopus (507) Google Scholar); this mask was then used to refine the NCS operators by maximizing the correlation between the electron density maps of the 4 molecules in the asymmetric unit using the program IMP (17Kleywegt G.J. Jones T.A. Acta Crystallogr. Sec. D. 1996; 52: 842-857Crossref PubMed Scopus (507) Google Scholar). Iterative cycles of density averaging, improvement of the mask, and refinement of the NCS operators, along with solvent flattening and phase extension to 2.6 Å resolution, resulted in a map of interpretable quality. The model was traced using the program O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Nearly the complete polypeptide chain of one monomer could be interpreted in agreement with the amino acid sequence. By applying the refined NCS operators to the coordinates of the first molecule, coordinates for the other three molecules in the asymmetric unit were generated. The four molecules were then refined using the program X-PLOR (19Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, Connecticut1992Google Scholar). During the first runs of the refinement (simulated annealing and individual B-factor refinement), tight NCS restraints were applied (17Kleywegt G.J. Jones T.A. Acta Crystallogr. Sec. D. 1996; 52: 842-857Crossref PubMed Scopus (507) Google Scholar), but in the final stage of the refinement (conventional positional refinement and individual B-factor refinement), they were gradually released or not even used at all for those residues that clearly showed different conformations in the 4 monomers in the asymmetric unit. The best refinement results were obtained using a flat bulk solvent correction. Special care was taken in the selection of the test set for theR free calculation; the test set was selected by dividing the reflections in 102 thin-resolution shells to minimize the correlation between test set and working set reflections that could be caused by the presence of NCS (20Kleywegt G.J. Jones T.A. Structure (Lond.). 1995; 15: 535-540Abstract Full Text Full Text PDF Scopus (224) Google Scholar). Water molecules were placed according to strict density and distance criteria, starting with the buried and NCS-related ones. The final model consists of 4 × 282 residues, 610 water molecules (33 of them refined with double positions), and 4 potassium ions. The crystallographic R factor andR free are 19.0% and 22.7%, respectively. PROCHECK (21Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHATCHECK (22Hooft R.W.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1788) Google Scholar) were used to assess the stereochemical quality. The structure was further analyzed using the program VOIDOO (23Kleywegt G.J. Jones T.A. Acta Crystallogr. Sec. D. 1994; 50: 178-185Crossref PubMed Scopus (970) Google Scholar), the programs from the CCP4 suite (14Collaborative Computational Project Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar), the BIOMOL package, and the program DALI (24Holm L. Sander C. Proteins. 1994; 3: 165-173Crossref Scopus (239) Google Scholar). Refinement statistics are given in TableII. The atomic coordinates and the structure factors have been deposited to the Protein Data Bank with the entry code 1ehy.Table IIRefinement statistics and stereochemical quality of the final modelResolution range (Å)25–2.1RfactoraR factor = ∑h∥F obs‖ − ‖F calc∥/∑‖F obs‖, whereF obs and F calc are the observed and calculated structure factor amplitudes, respectively. (R freebFree R-factor is calculated with 5% of the diffraction data selected randomly in 102 thin resolution shells that were not used during the refinement.)0.190 (0.227)No. of residues in the asymetric unit4 × 282No. of water molecules610No. of potassium ions4Average B-factor (Å2)Overall28.3Main chain27.2Side chain28.7Water molecules33.5Potassium ions33.3r.m.s. deviation from idealitybond lengths (Å)0.008bond angles (°)1.338Ramachandran plotResidues in most favored regions (%)89.7Residues in additional allowed regions (%)9.3Residues in generously allowed regions (%)1.0a R factor = ∑h∥F obs‖ − ‖F calc∥/∑‖F obs‖, whereF obs and F calc are the observed and calculated structure factor amplitudes, respectively.b Free R-factor is calculated with 5% of the diffraction data selected randomly in 102 thin resolution shells that were not used during the refinement. Open table in a new tab As a starting model, the atomic coordinates of the refined structure of the wild type epoxide hydrolase were used in which only the internal solvent molecules were retained. The crystal structure was energy-minimized prior to the modeling using a conjugate gradient routine implemented in X-PLOR (19Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, Connecticut1992Google Scholar). To completely remove the possible bias because of the conformation of the protein in the crystal, a slow-cooling molecular dynamics simulation (19Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, Connecticut1992Google Scholar) of 25 ps with temperature coupling (25Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Di Nola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (22550) Google Scholar) was performed in which the temperature was slowly reduced from 1000 K to 300 K. The missing loop 138–148 and the loop containing Asp246 were built using the program O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). To model a likely conformation of the active Ephy enzyme, we assumed that the acid member of the catalytic triad, Asp246, should lie at interaction distance to the catalytic His275 side chain, as found in many other members of the α/β hydrolase-fold family (9Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1821) Google Scholar). Haloalkane dehalogenase (PDB accession code 2HAD) (26Verschueren K.H.G. Franken S.M. Rozeboom H.J. Kalk K.H. Dijkstra B.W. J. Mol. Biol. 1993; 232: 856-872Crossref PubMed Scopus (128) Google Scholar) and bromoperoxidase A2 (PDB accession code1BRO) (27Hecht H.J. Sobek H. Haag T. Pfeifer O. van Pée K.-H. Nat. Struct. Biol. 1994; 1: 532-537Crossref PubMed Scopus (113) Google Scholar) were used as templates to model the new Ephy Asp246 position, analogous to Asp260 of dehalogenase and Asp228 of bromoperoxidase, respectively. Secondly, we assumed that the Gln134 side chain should be removed from the active site, as it blocks the putative substrate binding site. This was done by giving the Pro132-Ile133-Gln134 loop a similar conformation as the human pancreatic lipase (PDB accession code 1LPB) (28Egloff M.P. Marguet F. Buono G. Verger R. Cambillau C. van Tilbeurgh H. Biochemistry. 1995; 34: 2751-2762Crossref PubMed Scopus (254) Google Scholar) Pro177-Ala178-Glu179 motif, which has an equivalent topological position. The loop of residues 138–148, which is not observed in the electron density, was built like in the bromoperoxidase structure, connecting the core and the cap domains. Several cycles of stereochemical regularization were performed using the REFI and LEGO option of O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). The model was subsequently subjected to energy minimization to tidy up unacceptable close contacts and poor stereochemistry. To overcome the possibility that the energy-minimized structure was trapped in a local minimum, a second molecular dynamics-simulated annealing run was performed using the same setting as before. Extensive energy minimization was applied until convergence was reached, leading to a model with no residues outside the allowed regions in the Ramachandran plot (29Ramakrishnan C. Ramachandran G.N. J. Mol. Biol. 1965; 7: 95-99Google Scholar) and good stereochemical quality (r.m.s. deviation bond lengths = 0.005 Å, r.m.s. deviation bond angles = 1.61°). Asp107 and Asp131 have slightly deviating backbone torsion angles, like in the x-ray structure. Although the position of the modeled loop 132–148 is only one of the possible conformations it can assume, we are confident that the rebuilding of the loop containing Asp246, in a fashion common to many α/β hydrolase-fold enzymes, gives a plausible picture of the catalytic site of the fully active epoxide hydrolase. Epoxide hydrolase from A. radiobacter AD1 (Ephy) crystallizes in the monoclinic space groupC2, with 4 molecules in the asymmetric unit. A superimposition of the Cα atoms of the four molecules gave an average r.m.s. difference of 0.24 Å for molecules B, C, and D and a higher r.m.s. difference of 0.40 Å if molecule A is included. All results discussed below apply to all 4 molecules (A, B, C, and D) unless stated otherwise. The final model consists of 4 × 282 residues, 610 water molecules (33 of them refined with double positions), and 4 potassium ions, originating from the crystallization buffer, one for each molecule in the asymmetric unit. In each monomer (294 residues), the first N-terminal residue (Met) is not visible nor is there interpretable electron density for the loop 138–148. The final crystallographic R factor and R freevalues are 19.0% and 22.7%, respectively. The r.m.s. deviations from ideal geometry are 0.008 Å for bond lengths and 1.338° for bond angles. No residues are in the disallowed regions of the Ramachandran plot (29Ramakrishnan C. Ramachandran G.N. J. Mol. Biol. 1965; 7: 95-99Google Scholar). Pro39 was found in a cisconformation. The Ephy monomer has a nearly globular shape with approximate dimensions of 48 × 47 × 47 Å3. It consists of two domains: domain I (or “core” domain), which shows the typical features of the α/β hydrolase-fold topology (9Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1821) Google Scholar), and the mainly α-helical domain II (or “cap” domain), which lies on top of domain I (Figs.1 and 2). The core domain comprises amino acids 1–137 and 219–294, and it consists of a central eight-stranded β-sheet with seven parallel strands (only the second strand is antiparallel). The β-sheet is flanked on both sides by α-helices, two on one side and four on the other. Helix α9 is a one-turn 310 helix. Domain II, containing α-helices α4 to α8, forms a large excursion between β-strands β6 and β7 of the core domain. It has a double-layered structure with helices α7 and α8 located between the core domain and the plane formed by α4, α5, and α6.Figure 2Secondary structure topology diagram and location of the catalytic triad residues, Asp107, Asp246, and His275. The dashed line represents the missing loop 138–148. Short 310helices are located at the N terminus (α′), between β3 and α1 (α′1), between β4 and α2 (α′2), and between α3 and β6 (α′3). The last α-helix shows a conspicuous bend at residue 281 (α′11-α11) because of the presence of Pro282 in the center of the helix.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The proposed active-site residues (6Rink R. Fennema M. Smids M. Dehmel U. Janssen D.B. J. Biol. Chem. 1997; 272: 14650-14657Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) Asp107 and His275 are located in a predominantly hydrophobic internal cavity between domains I and II. The core domain contributes to the lining of the cavity with residues Gly37, Trp38, Pro39, Glu44, His106, Asp107, Phe108, Ile133, Phe137, Ile219, His275, Phe276, Val279. The cap domain supplies Tyr152, Trp183, and Tyr215 (Fig. 3). Asp107 is situated at the very sharp “nucleophile elbow” between the central strand β5 and helix α3. At this topological position, all α/β hydrolase-fold enzymes present the nucleophile, which can either be Ser, Cys, or Asp (9Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1821) Google Scholar). The (φ, ψ) angles of Asp107 are slightly unfavorable (φ = 57°, ψ = −124°), but its conformation is stabilized by a network of hydrogen bonds involving residues of the sharp turn, as has been found in other α/β hydrolase enzymes (9Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1821) Google Scholar). In addition, the main chain nitrogen atom of Asp107 interacts via a hydrogen bond with the backbone oxygen atom of Asp131, the other residue with slightly deviating backbone torsion angles (φ = 31°, ψ = 69°). Furthermore, the side chain of Asp107 is stabilized by a hydrogen bond of its Oδ2 atom with the backbone amide groups of Trp38 and Phe108 and by a salt bridge between the Oδ1 atom of Asp107 and the Nε2 atom of the His275 side chain. An ∼20-Å long tunnel, filled with water molecules, is located between α-helices α1, α10, the loop connecting α-helix α1 and β-strand β3 of the core domain, and α7 of the cap domain (Fig. 4). This tunnel leads to the back of the active-site cavity, and it is perfectly suited to replenish the hydrolytic water molecule at hydrogen bond distance to the Nε2 atom of the His275 side chain (Fig. 3) after the reaction. In our structure, the active site cavity is exposed to the solvent from the front part too, where the missing l" @default.
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• W2099381109 title "The X-ray Structure of Epoxide Hydrolase from Agrobacterium radiobacter AD1" @default.
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