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• W2010618393 abstract "Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75Å resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an α-β-β-α four-layer architecture characteristic of the nitrilase superfamily. One exterior α layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight α-β-β-α-α-β-β-α dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30Å, respectively. These structures reveal a small formamide-binding pocket that includes Cys166, Glu60, and Lys133 catalytic residues, in which Cys166 acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl d-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently. Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75Å resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an α-β-β-α four-layer architecture characteristic of the nitrilase superfamily. One exterior α layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight α-β-β-α-α-β-β-α dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30Å, respectively. These structures reveal a small formamide-binding pocket that includes Cys166, Glu60, and Lys133 catalytic residues, in which Cys166 acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl d-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently. Utilization of nitrogen from nonpeptide carbon-nitrogen compounds usually involves nitrilase or nitrile hydratase coupled with amidase to produce ammonia for further assimilation or transfer (1Willison J.C. FEMS Microbiol. Rev. 1993; 10: 1-38Crossref PubMed Google Scholar). Based on multiple sequence analysis, nitrilases, cyanide hydratases, and aliphatic amidases, along with β-alanine synthases that reduce organic nitrogen compounds are found to possess several conserved sequence features, including an invariant cysteine active residue (2Bork P. Koonin E.V. Protein Sci. 1994; 3: 1344-1346Crossref PubMed Scopus (98) Google Scholar). The first determined structure of the nitrilase (Nit) domain is from worm NitFhit that shows a tetramer, and each Nit domain has a four-layer α-β-β-α sandwich fold (3Pace H.C. Hodawadekar S.C. Draganescu A. Huang J. Bieganowski P. Pekarsky Y. Croce C.M. Brenner C. Curr. Biol. 2000; 10: 907-917Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Additionally, a novel Cys-Glu-Lys (CEK) triad that contains the highly conserved cysteine is seen in a solvent-accessible pocket. Crystal structures of N-carbamoyl d-amino acid amidohydrolase reveal a homologous fold, in which the CEK triad is also found (4Nakai T. Hasegawa T. Yamashita E. Yamamoto M. Kumasaka T. Ueki T. Nanba H. Ikinaka Y. Takahashi S. Sato M. Tsukihara T. Structure. 2000; 8: 729-739Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (71) Google Scholar). Structural and site-directed mutagenesis results suggest the roles of the CEK residues; the active cysteine acts as the nucleophile, glutamate mediates the proton transfer, and lysine stabilizes a tetrahedral transition state (6Chen C.Y. Chiu W.C. Liu J.S. Hsu W.H. Wang W.C. J. Biol. Chem. 2003; 278: 26194-26201Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 7Grifantini R. Pratesi C. Galli G. Grandi G. J. Biol. Chem. 1996; 271: 9326-9331Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Such a novel catalytic triad had not been observed in any other hydrolytic enzymes. In 2001, Pace and Brenner (8Pace H.C. Brenner C. Genome Biol. 2001; 2: 1-9Crossref Google Scholar) defined these proteins having the strictly conserved CEK site as the nitrilase superfamily. On the basis of sequence similarity and the presence of additional domains, 13 branches can be classified, nine of which have known or deduced specificity for specific nitrile or amide hydrolysis or amide condensation reactions, including nitrilase, aliphatic amidase, N-terminal amidase, biotinidase, β-ureidopropionase, carbamoylase, glutaminase domain of glutaminedependent NAD synthetase, and apolipoprotein N-acyltransferase. Subsequently, structures from two members of the nitrilase superfamily (a yeast hypothetical protein with sequence homologous to CN hydralase from yeast (9Kumaran D. Eswaramoorthy S. Gerchman S.E. Kycia H. Studier F.W. Swaminathan S. Proteins. 2003; 52: 283-291Crossref PubMed Scopus (47) Google Scholar) and a hypothetical protein from Pyrococcus horikoshii (10Sakai N. Tajika Y. Yao M. Watanabe N. Tanaka I. Proteins. 2004; 57: 869-873Crossref PubMed Scopus (35) Google Scholar)) show the characteristic α-β-β-α sandwich architecture. Given the functional diversity and the wide evolutionary distribution of the nitrilase superfamily members, we sought to investigate the structure-function relationship of Helicobacter pylori formamidase AmiF that shows restricted substrate specificity. H. pylori is a human gastric pathogen that persistently colonizes the mucus layer overlaying the epithelium of the stomach. AmiF and the other paralogue AmiE, along with urease, play a major role in producing ammonia to protect against gastric acidity or as a nitrogen source or a cytotoxic molecule, enabling H. pylori to adapt into such an exclusive environment (11Skouloubris S. Labigne A. De Reuse H. Mol. Microbiol. 1997; 25: 989-998Crossref PubMed Scopus (89) Google Scholar, 12Skouloubris S. Labigne A. De Reuse H. Mol. Microbiol. 2001; 40: 596-609Crossref PubMed Scopus (67) Google Scholar). In response to growth at mild acidic conditions, genes encoding these proteins are up-regulated (13van Vliet A.H. Kuipers E.J. Stoof J. Poppelaars S.W. Kusters J.G. Infect. Immun. 2004; 72: 766-773Crossref PubMed Scopus (78) Google Scholar, 14Bury-Mone S. Thiberge J.M. Contreras M. Maitournam A. Labigne A. De Reuse H. Mol. Microbiol. 2004; 53: 623-638Crossref PubMed Scopus (166) Google Scholar). Both AmiF and AmiE belong to members of aliphatic amidases. The presumed invariant active cysteine is identified as Cys165 for AmiE and Cys166 for AmiF from site-directed mutagenesis studies (12Skouloubris S. Labigne A. De Reuse H. Mol. Microbiol. 2001; 40: 596-609Crossref PubMed Scopus (67) Google Scholar). Additionally, AmiF is noted to demonstrate restricted substrate specificity in which formamide is its only known substrate. Here we report the 1.75 Å crystal structure of the native AmiF. Crystal structures of an inactive mutant AmiF C166S were also determined in its apo and liganded forms. Structural analysis reveals a small binding pocket that has the key CEK catalytic residues to reduce the organic nitrogen compounds efficiently. Cloning, Site-directed Mutagenesis, Expression, and Purification—The gene (amiF) encoding formamidase was amplified from chromosomal DNA of H. pylori 26695 by PCR using pfu DNA polymerase and inserted into the pQE30 expression vector to generate pQE30-AmiF. Primers (AmiF-F, 5′-CGCGGATCCATGGGAAGTATCGGTAGT-3′); AmiF-R, 5′-CCCAAGCTTGGGTTATTTCCCAAAACG-3′) that contain sequences for the BamHI site and HindIII site, respectively, were designed based on the nucleotide sequence of the reported amiF gene of H. pylori 26695 (accession number NC_000915) (15Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzegerald L.M. Lee N. Adams M.D. Hickey E.K. Berg D.E. Gocayne J.D. Utterback T.R. Peterson J.D. Kelley J.M. Cotton M.D. Weidman J.M. Fujii C. Bowman C. Watthey L. Wallin E. Hayes W.S. Borodovsky M. Karp P.D. Smith H.O. Fraser C.M. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3028) Google Scholar). Site-directed mutagenesis was performed essentially using the method of overlap extension PCR with plasmid pQE30-AmiF as the template (16Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar, 17Chiu W.C. You J.Y. Liu J.S. Hsu S.K. Hsu W.H. Shih C.H. Hwang J.K. Wang W.C. J. Mol. Biol. 2006; 359: 741-753Crossref PubMed Scopus (29) Google Scholar). All mutations were confirmed by sequencing of the whole ligated PCR fragment. Expression of wild-type AmiF or the C166S mutant in Escherichia coli JM109 cells transformed with pQE30-AmiF or pQE30-C166S was induced at 37 °C. Bacterial pellets were fractionated, and soluble proteins in cytosolic fractions were collected. The expressed protein with a His6 tag was purified by immobilized nickel-ion chromatography, followed by MonoQ ion exchange chromatography (GE Healthcare), and then analyzed by SDS-PAGE to verify the purity. The protein concentration was assayed according to the Bradford method with bovine serum albumin as a standard (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Expression of selenomethionine-labeled AmiF (Se-Met-AmiF) 3The abbreviations used are: Se-Met, selenomethionine; Nit, nitrilase; PEG, polyethylene glycol; GNM, Gaussian network model; r.m.s., root mean square; MME, monomethyl ether. was done essentially described by Yang et al. (19Yang W. Hendrickson W.A. Kalman E.T. Crouch R.J. J. Biol. Chem. 1990; 265: 13553-13559Abstract Full Text PDF PubMed Google Scholar) and Van Duyne et al. (20Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1091) Google Scholar). In brief, an overnight culture of transformed E. coli JM109 was grown in 1 liter of LB medium and 100 mg/liter ampicillin. The cells from the overnight culture were harvested and resuspended in 1 liter of M9 medium (12.8 g/liter Na2HPO4, 3 g/liter K2HPO4, 0.5 g/liter NaCl, 1 g/liter NH4Cl). Twenty ml of M9-add (20% glucose, 1 m MgSO4, 1 m CaCl2, 0.5% thiamine, 100 mg/liter ampicillin) were then added per liter of culture. When the absorbance at 600 nm reached A600 nm = 0.6, 10 ml of Met-shutdown mix (100 mg/liter lysine hydrochloride, 100 mg/liter threonine, 100 mg/liter phenylalanine, 50 mg/liter leucine, 50 mg/liter isoleucine, 50 mg/liter valine, 120 mg/liter seleno-l-methionine) were added per liter of culture. The culture was induced by isopropyl 1-thio-β-dgalactopyranoside and incubated overnight at 37 °C. Purification of Se-Met-AmiF was performed based on the procedures for the native AmiF. Crystallization—All crystallization was performed by the hanging drop vapor diffusion method at 20 °C. Initial crystallization conditions were screened using Crystal Screen I and II kits (Hampton Research) and Clear Strategy Screen I and II kits (Molecular Dimension). One μl of protein solution (10 mg/ml) in 50 mm Tris-HCl, pH 8.0, was mixed with an equal volume of the well precipitant. The best diffracting AmiF crystals were found in a modified solution containing 0.1 m sodium cacodylate (pH 6.5), 0.15 m potassium thiocynate, and 20% polyethylene glycol (PEG) 550 MME. The crystal grew as rods and reached a maximum size of about 0.6 × 0.1 × 0.1 mm within 2 days at 20 °C. The native crystal was characterized as belonging to space group R3, with the unit cell dimensions a = b = 147.21, c = 72.27 Å. There are two molecules per asymmetric unit. Crystals of the Se-Met-AmiF protein were obtained in a condition (0.1 m sodium cacodylate (pH 6.0), 0.8 m sodium formate, 8% PEG 20,000, and PEG 550 MME) using Se-Met-AmiF (20 mg/ml). Rod-shaped crystals with a maximum size of about 0.6 × 0.1 × 0.1 mm grew within 4 days at 20 °C. The Se-Met-AmiF crystal also belongs to space group R3 with unit cell dimensions of a = b = 147.80 Å and c = 72.78 Å. Crystals of C166S were obtained in a solution containing 0.1 m sodium acetate (pH 5.0), 0.2 m lithium sulfate, and 14% PEG 2000 MME. Of two crystal forms (thin plates or rods), only the thin plate crystal diffracted beyond 3 Å using the in-house x-ray source. The thin plate crystal was characterized as space group C2, with the unit cell dimensions of a = 117.72, b = 130.53, c = 144.59 Å, and β = 99.44°. There are six molecules per asymmetric unit. Attempts to obtain liganded C166S crystals using the soaking method failed. After extensive trials, the C166S complex crystal was finally obtained by the co-crystallization method in a solution containing 0.1 m sodium acetate (pH 5.0), 0.2 m lithium sulfate, 6% PEG 1500, and 3 mm formamide. The complex crystal grew as a rod and was characterized as the space group P21 with cell dimensions of a = 83.09, b = 151.80, c = 89.08 Å, and β = 114.99°. There are six molecules per asymmetric unit. Data Collection—Prior to data collection, crystals were dipped into Fomblin® cryoprotectant oil for several seconds and then flash-frozen in a liquid nitrogen stream. Diffraction data were collected using a MSC X-Stream cryosystem and an R-AXIS IV++ image plate system with double mirror-focused CuKα x-ray radiation generated from a Rigaku RU-300 rotating anode generator at the Macromolecular X-ray Crystallographic Laboratory of National Tsing Hua University, Hsinchu, Taiwan. The 1.75 Å native data set, the 2.27 Å Se-Met-AmiF data set, the 2.50 Å C166S data set, and the 2.30 Å C166S complex data set were collected on the BL12B2 Taiwan beamline at SPring-8, Japan, using an ADSC Quantum 4R CCD detector. All data sets were collected at -165 °C and processed with the HKL/HKL2000 suite (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Data collection statistics are shown in Table 1.TABLE 1Crystallographic dataParametersValuesAmiFC166SNative AmiFSe-Met-AmiFC166SC166S complexData collection and phasingSpace groupR3R3C2P21Cell dimensionsa (Å)147.21147.80117.7283.09b (Å)147.21147.80130.53151.80c (Å)72.2772.78144.5989.08β = 99.4°β = 115.0°Wavelength (Å)0.98000.97910.95371.0000Resolution (Å)30.0-1.7530.0-2.2730.0-2.5030.0-2.30Highest resolution shell (Å)1.81-1.752.35-2.272.59-2.502.38-2.30Completeness (%)aValues in parentheses refer to statistics in the highest resolution shell.99.7 (100.0)99.1 (95.8)99.8 (99.8)91.4 (92.5)Average I/σ (I)15.2 (2.7)10.7 (2.7)8.4 (4.6)18.0 (12.4)Number of unique reflections55,50070,94277,740Redundancy3.22.53.33.4Rmerge (%)bRmerge = ∑|Iobs - 〈I 〉|/∑Iobs.7.8 (49.8)6.5 (31.0)3.6 (10.0)5.4 (35.4)Overall figure of merit (acentric/centric)cFigure of merit = |Fbest|/|F|.0.68/0.87Solvent content (%)39.851.146.1RefinementResolution range (Å)30.0-1.7530.0-2.5030.0-2.30Number of atomsProtein509814,90115,007Solvent713850735Ligand9Average B-factor (Å2)Protein21.89.536.5Water36.19.735.8Ligands41.1R factordR = ∑|Fo - Fc|/∑Fo, where Fo and Fc are the observed and calculated structure-factor amplitudes, respectively.0.1740.2580.253RfreeeRfree was computed using 5% of the data assigned randomly.0.2150.2940.285r.m.s. deviation bond length (Å)0.0060.0040.006r.m.s. deviation bond angles (degrees)1.0370.8820.947Estimated coordinate error (Å)0.0830.1980.228Ramachandran analysis (%)fEstimated standard uncertainties based on maximum likelihood.Favored/Allowed/Generous/Disallowed88.2/10.6/0.7/0.484.3/13.6/1.7/0.489.0/10.3/0.4/0.4a Values in parentheses refer to statistics in the highest resolution shell.b Rmerge = ∑|Iobs - 〈I 〉|/∑Iobs.c Figure of merit = |Fbest|/|F|.d R = ∑|Fo - Fc|/∑Fo, where Fo and Fc are the observed and calculated structure-factor amplitudes, respectively.e Rfree was computed using 5% of the data assigned randomly.f Estimated standard uncertainties based on maximum likelihood. Open table in a new tab Structure Determination and Refinement—The AmiF structure was solved by single wavelength anomalous dispersion methods. Twelve selenium sites were located with the program SOLVE (22Terwilliger T.C. Berendzen J. Acta. Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Phase determination, density modification, and automated model building with SOLVE/RESOLVE (22Terwilliger T.C. Berendzen J. Acta. Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar, 23Terwilliger T.C. Acta. Crystallogr. Sect. D. 2000; 56: 965-972Crossref PubMed Scopus (1636) Google Scholar) led to an interpretable density map and initial model. The atomic model was further built using O (24Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta. Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Crystallographic refinement was carried out using the maximum likelihood target function embedded in program REFMAC5 (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta. Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar, 26Collaborative Computational Project 4Acta. Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The native AmiF model was obtained using MOLREP (27Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) and refined using REFMAC5 (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta. Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) coupled to ARP/wARP (28Lamzin V. Wilson K.S. Acta. Crystallogr. Sect. D. 1993; 49: 127-147Crossref Google Scholar), which was used to add water molecules automatically. The protein model was assessed using the program PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The apo and liganded C166S structures were determined by the molecular replacement methods using the native AmiF structure as a search model by MOLREP (27Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) and refined by REFMAC5 (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta. Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) coupled to ARP/wARP (28Lamzin V. Wilson K.S. Acta. Crystallogr. Sect. D. 1993; 49: 127-147Crossref Google Scholar). Structural Comparisons—Structure comparisons with N-carbamoyl-d-amino acid amidohydrolase (Protein Data Bank entry codes 1ERZ and 1FO6) (4Nakai T. Hasegawa T. Yamashita E. Yamamoto M. Kumasaka T. Ueki T. Nanba H. Ikinaka Y. Takahashi S. Sato M. Tsukihara T. Structure. 2000; 8: 729-739Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (71) Google Scholar), the Nit domain of NitFhit (Protein Data Bank entry code 1EMS) (3Pace H.C. Hodawadekar S.C. Draganescu A. Huang J. Bieganowski P. Pekarsky Y. Croce C.M. Brenner C. Curr. Biol. 2000; 10: 907-917Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and the putative CN hydrolase from yeast (Protein Data Bank entry code 1F89) (10Sakai N. Tajika Y. Yao M. Watanabe N. Tanaka I. Proteins. 2004; 57: 869-873Crossref PubMed Scopus (35) Google Scholar), were carried out using the program LSQMAN in O (24Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta. Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) to superimpose Cα atoms. Combined sequence and secondary structure alignments and figure preparation (Fig. 5A) were done with the program ESPript (30Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2540) Google Scholar). Structural figures (Figs. 1, 2A, 3, 4, and 6) were prepared with the program PyMol (42DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar) (available on the World Wide Web at www.pymol.org). Fig. 5B was prepared with the programs MOLSCRIPT (31Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and RASTER3D (32Merrit E.A. Murphy M.E.P. Acta. Crystallogr. Sect. D. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar).FIGURE 1Structure of AmiF. A, ribbon representation of the hexameric AmiF structure (AB)3 viewed down the 3-fold axis. Three A subunits (A1, A2, and A3) are depicted in green, and three B subunits (B1, B2, and B3) are in red. B, ribbon representation of the dimeric AmiF structure (AB) viewed down a 2-fold axis. E60 (red), K133 (blue), and C166 (orange) are drawn as stick models. The oxygen, nitrogen, and sulfur atoms are colored in red, dark blue, and yellow, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2A, stereoview of the monomeric AmiF. Glu60 (red), Lys133 (blue), and Cys166 (orange) are drawn as stick models. The oxygen, nitrogen, and sulfur atoms are colored in red, dark blue, and yellow, respectively. B, topology of the monomeric AmiF.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3The 2Fo - Fc electron density map of the inactive mutant C166S in its apo form (A) and model 1 (B) and model 2 (C) of the liganded C166S complex around C166S, Glu60, and Lys133, contoured at the 1.0-σ level. The bound formamide (FRA) is drawn as ball-and-stick models. The carbon, oxygen, and nitrogen atoms are colored in white, red, and blue, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4The binding pocket of the C166S·formamide complex. A, stereoview of the C166S·formamide binding pocket. Loops (β2-η1-α2 (residues 58-82), β5-β6 (residues 133-152), and β8-α4 (residues 191-197)) and a short 310 turn between β7 and α3 (residues 166-170) enclosing formamide are colored beige. Glu60, Lys133, and C166S are shown as heavy gray sticks, whereas other ligand-binding residues are shown as green sticks. B, superposition between apo-C166S (green) and C166S·formamide (orange) subunits. C166S-Glu60-Lys133 residues are shown as heavy sticks. Formamide (FRA) in A and B is drawn as ball-and-stick models. The carbon, oxygen, and nitrogen atoms are colored white, red, and blue, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6A, superposition of the binding sites between AmiF (C166S)·formamide (orange) and N-carbamoyl d-amino acid amidohydrolase (C172S)·N-carbamoyl-d-p-hydroxyphenylglycine (HPG)(green) complex structures. Catalytic CEK residues are shown as heavy sticks, whereas other ligand-binding residues are shown as thin sticks. Formamide (FRA) and hydroxyphenylglycine are drawn as ball-and-stick models. The carbon, oxygen, nitrogen, and sulfur atoms are colored white, red, blue, and yellow, respectively. B, molecular surfaces of AmiF (C166S)·FRA (left) and N-carbamoyl d-amino acid amidohydrolase (C172S)·HPG (right) complexes. Surfaces are colored by the type of residues: glutamate (red), cysteine (orange), lysine (blue), histidine (cyan), arginine (dark blue); serine (yellow orange), asparagine (green), threonine (cyan), tryptophan (brown), and tyrosine (yellow). FRA and HPG are drawn as ball-and-stick models. The carbon, oxygen, and nitrogen atoms are colored white, red, and blue, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Molecular Dynamic Simulations and Gaussian Network Model—Dynamic simulations were performed with the GROMACS 3.3 program running on a Fedora Linux system essentially as previously described (17Chiu W.C. You J.Y. Liu J.S. Hsu S.K. Hsu W.H. Shih C.H. Hwang J.K. Wang W.C. J. Mol. Biol. 2006; 359: 741-753Crossref PubMed Scopus (29) Google Scholar, 33Berendsen H.J.C. v. d. S.D. van Drunen R. Comp. Phys. Commun. 1995; 91: 43-56Crossref Scopus (7382) Google Scholar). All simulations were carried out for 2 ns at 300 K. The first 1-ns run was used to attain equilibrium, whereas the trajectories of the last 1 ns were used for analysis. The low frequency motions were calculated based on the Gaussian network model (GNM) (34Bahar I. Atilgan A.R. Erman B. Fold Des. 1997; 2: 173-181Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar, 35Eichinger B.E. Macromolecules. 1972; 5: 496-505Crossref Scopus (97) Google Scholar). Structure Description of the Native AmiF—The 1.75 Å resolution electron density map of AmiF revealed two molecules (AB) per asymmetric unit. Except for the N-terminal segment (residues 1-12) that contained weak or negative density and could not be built into the model, the main-chain and sidechain atoms from 13 to 334 residues are well defined in A and B subunits. Only one residue, Cys166, lies in the disallowed region of the Ramachandran plot, despite its low B factor. The unusual geometry is stabilized by three hydrogen bonds around this peptide unit: Cys166 O···N Asp168, Cys166 O···N Gly169, and Cys166 O···Nη2 Arg188. The final model was refined to an R value of 17.4% (Rfree = 21.5%; Table 1). The average B-factor for all polypeptide atoms is 23.6 Å2. In the crystal structure, AmiF is a hexamer (Fig. 1): Subunit A and Subunit B associate into an AB dimer related by a noncrystallographic 2-fold axis (Fig. 1B). Along the 3-fold axis, three AB dimers assemble into a hexamer (A1B1A2B2A3B3) (Fig. 1A). The overall shape of each trimer (A1A2A3 or B1B2B3) is approximately an equilateral triangular prism (∼95 Å along the prism axis). Each monomer folds into a four-layer α-β-β-α structure, characteristic of members in the nitrilase superfamily (Fig. 2, A and B). One exterior α layer (α1 and α2) is solvent-accessible to shield the inner β sheets from the solvent, whereas the other α layer (α3-α7) associates with the neighboring Subunit B. One side of the four-layer sandwich contains short turns or loops to connect the secondary elements, whereas the other side has several long loops, particularly the β2-η1-α2, β5-β6, and β9-β10 segments. Buried residues at the A-B intersubunit interface are largely from residues of the β5-β6 loop, near or in those of α3-α7 helices and the C-terminal region of each monomer. The symmetric assembly (α3 versus α3, α4 versus α4, and α7 versus α7) forms a tightly associated α-β-β-α-α-β-β-α dimer (Fig. 1B). In total, there are 456 interactions within 3.8 Å, including 36 hydrogen bonds and 13 salt bridges. The total solvent-accessible surface of the AB dimer is ∼29,700 Å2, calculated by protein interfaces, surfaces, and assemblies service PISA at the European Bioinformatics Institute (available on the World Wide Web at www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Of the subunit surface area, 28.8% is used for the formation of the dimer. Crystal Structures of C166S—To obtain liganded structures, crystallization trials using the co-crystallization or soaking methods in the presence of various concentrations of formamide were attempted. Of various structures, only one showed additional nonpeptide electron density near Cys166 (data not shown). Given the relatively large peak, either formamide or a solvent cacodylate molecule could be docked into this density in either subunit. Nonetheless, a piece of residual density that could not be ignored is present in either model, indicating a possibility that formamide and cacodylate co-exist in this density. However, we were not able to model both properly into this density, possibly due to the disordered formamide/cacodylate displacement. To further clarify the catalytic site, we have obtained the crystal structure of an inactive mutant, C166S, in a crystallization condition without cacodylate. The model consists of six subunits. Like the wild-type structure, the N-terminal segment (residues 1-12) is not visible in each subunit. Disordered regions that could not be defined include the 283-288 region and residues in loops (A194, A249, A299, B194, B249, B298, C194, C226, C300, D194, D232, D248, D296, D298, E194, E298, F194, and F248). Crystallographic data are given in Table 1. Crystal Structure of C166S·Formamide Complex—After extensive trials, the complex crystal was also obtained by the co-crystallization method in a solution containing 3 mm formamide. The structure was" @default.
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• W2010618393 title "Crystal Structure of Helicobacter pylori Formamidase AmiF Reveals a Cysteine-Glutamate-Lysine Catalytic Triad" @default.
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