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• W1966354566 abstract "Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 Å resolution with finalR-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal α-helical and C-terminal β-sheet domains, which constitute incomplete α5/α5-barrel and anti-parallel β-sheet structures, respectively. A deep cleft is located in the N-terminal α-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg313 and Tyr315 residues in the loop from the N-terminal domain and the Arg612 residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme. Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 Å resolution with finalR-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal α-helical and C-terminal β-sheet domains, which constitute incomplete α5/α5-barrel and anti-parallel β-sheet structures, respectively. A deep cleft is located in the N-terminal α-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg313 and Tyr315 residues in the loop from the N-terminal domain and the Arg612 residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme. pyruvylated mannose d-glucose d-glucuronic acid d-mannose N-acetyl-d-glucosamine N-acetyl-d-galactosamine multiple isomorphous replacement amino acid root-mean-square water molecule acetate potassium phosphate buffer N,N-bis(2-hydroxyethyl)glycine crystallography NMR software protein data bank There is a large number of polysaccharide-degrading enzymes. 1B. Henrissat, P. Coutinho, and E. Deleury, afmb.cnrs-mrs.fr/∼cazy/CAZY/index.html. 1B. Henrissat, P. Coutinho, and E. Deleury, afmb.cnrs-mrs.fr/∼cazy/CAZY/index.html. Generally, they can be classified into two groups, hydrolases and lyases. The former catalyze the hydrolysis reaction responsible for breaking glycosidic bonds in polysaccharides. The properties of glycosyl hydrolases that act on poly- and oligosaccharides have been well documented, and the three-dimensional structures of many polysaccharide hydrolases, such as amylases, chitinases, and cellulases, have already been reviewed (1Bourne Y. Henrissat B. Curr. Opin. Struct. Biol. 2001; 11: 593-600Crossref PubMed Scopus (355) Google Scholar, 2Davies G.J. Henrissat B. Biochem. Soc. Trans. 2002; 30: 291-297Crossref PubMed Google Scholar). As regards the second group, the lyases, it is known that they recognize uronic acid residues in polysaccharides, catalyze the β-elimination reaction, and produce unsaturated saccharides with C=C double bonds at the nonreducing terminal uronate residues (Fig. 1). These characteristics of lyases indicate that they share common structural features determining their uronate recognition sites and reaction modes (β-elimination reaction). Although structural analyses of lyases for pectate (3Yoder M.D. Keen N.T. Jurnak F. Science. 1993; 260: 1503-1507Crossref PubMed Scopus (397) Google Scholar, 4Yoder M.D. Lietzke S.E. Jurnak F. Structure. 1993; 1: 241-251Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 5Pickersgill R. Jenkins J. Harris G. Nasser W. Robert-Baudouy J. Nat. Struct. Biol. 1994; 1: 717-723Crossref PubMed Scopus (186) Google Scholar, 6Mayans O. Scott M. Connerton I. Gravesen T. Benen J. Visser J. Pickersgill R. Jenkins J. Structure. 1997; 15: 677-689Abstract Full Text Full Text PDF Scopus (164) Google Scholar, 7Vitali J. Schick B. Kester H.C. Visser J. Jurnak F. Plant Physiol. 1998; 116: 69-80Crossref PubMed Scopus (87) Google Scholar, 8Akita M. Suzuki A. Kobayashi T. Ito S. Yamane T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1786-1792Crossref PubMed Scopus (48) Google Scholar), alginate (9Yoon H.-J. Mikami B. Hashimoto W. Murata K. J. Mol. Biol. 1999; 290: 505-514Crossref PubMed Scopus (97) Google Scholar), hyaluronate (10Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 15: 1228-1240Crossref Scopus (145) Google Scholar, 11Li S. Jedrzejas M.J. J. Biol. Chem. 2001; 276: 41407-41416Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and chondroitin (12Huang W. Matte A. Li Y. Kim Y.S. Linhardt R.J. Su H. Cygler M. J. Mol. Biol. 1999; 294: 1257-1269Crossref PubMed Scopus (96) Google Scholar, 13Féthiere J. Eggimann B. Cygler M. J. Mol. Biol. 1999; 14: 635-647Crossref Scopus (88) Google Scholar) have been made, there is little information regarding the structural rules common to polysaccharide lyases. To determine the structural and functional relationships exhibited by polysaccharide lyases, we have recently been focusing on bacterial heteropolysaccharide lyases (lyases for alginate (14Yoon H.-J. Hashimoto W. Miyake O. Okamoto M. Mikami B. Murata K. Protein Expression Purif. 2000; 19: 84-90Crossref PubMed Scopus (95) Google Scholar), gellan (15Hashimoto W. Sato N. Kimura S. Murata K. Arch. Biochem. Biophys. 1998; 354: 31-39Crossref PubMed Scopus (27) Google Scholar), and xanthan (16Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 1998; 64: 3765-3768Crossref PubMed Google Scholar)) with either an endotypic or exotypic reaction mode and with either a backbone or side chain type of cleavage site. We have already determined the crystal structure of the endotype alginate lyase from Sphingomonas sp. A1 (9Yoon H.-J. Mikami B. Hashimoto W. Murata K. J. Mol. Biol. 1999; 290: 505-514Crossref PubMed Scopus (97) Google Scholar). Xanthan is an exopolysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris (17Rogovin S.P. Anderson R.F. Cadmus M.C. J. Biochem. Microbiol. Technol. Eng. 1961; 3: 51-63Crossref Google Scholar). This exopolysaccharide consists of a main cellulosic chain with trisaccharide side chains composed of one glucuronyl and two mannosyl residues attached at the C-3 position of alternate glucosyl residues (18Jansson P.-E. Kenne L. Lindberg B. Carbohydr. Res. 1975; 45: 275-282Crossref PubMed Scopus (645) Google Scholar) (Fig. 1 A). The internal and terminal mannosyl residues of the side chains have an O-acetyl group at the C-6 position and a pyruvate ketal at the C-4 and C-6 positions, respectively, although the extents of acetylation and pyruvation vary with the growth conditions and bacterial strain (19Sandford P.A. Pittsley J.E. Knutson C.A. Watson P.R. Cadmus M.C. Janes A. Sandford P.A. Laskin A. Extracellular Microbial Polysaccharides Symposium Series No. 45. American Chemical Society, Washington, D. C.1977: 192-210Google Scholar). Because the polymer has the peculiar rheological properties of pseudoplasticity (reversible decrease in viscosity with increase in shear rate), high viscosity at low concentrations, and tolerance to a wide range of pH and temperatures, it is widely utilized as a gelling and stabilizing agent in the food, pharmaceutical, and oil industries (20Becker A. Katzen F. Puhler A. Ielpi L. Appl. Microbiol. Biotechnol. 1998; 50: 145-152Crossref PubMed Scopus (295) Google Scholar). Xanthan lyase produced by Bacillus sp. GL1 acts exolytically on the side chains of xanthan and liberates pyruvylated mannose (PyrMan)2 through the β-elimination reaction (Fig.1 A) (16Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 1998; 64: 3765-3768Crossref PubMed Google Scholar). The enzyme is synthesized as a precursor form (99 kDa) and is then converted into the mature form (∼75 kDa) through posttranslational excision of the signal peptide (2 kDa) and C-terminal polypeptide (∼22 kDa) (21Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 2001; 67: 713-720Crossref PubMed Scopus (23) Google Scholar). On the basis of amino acid (aa) sequence similarity, the enzyme is classified into polysaccharide lyase family 8,1 which contains lyases for hyaluronate and chondroitin AC in addition to xanthan lyase, although xanthan lyase does not act on hyaluronate and chondroitin (21Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 2001; 67: 713-720Crossref PubMed Scopus (23) Google Scholar). Moreover, xanthan lyase is peculiar in that it acts on the side chains of a polysaccharide and releases the nonreducing terminal saccharides of the side chains, because almost all polysaccharide lyases (including those for pectate, alginate, hyaluronate, chondroitin, and heparin) endolytically cleave the glycosidic bonds in the main chains of polysaccharides. Therefore, it is thought that the structural analysis of xanthan lyase will contribute to clarification of the structural features that determine the uronate recognition site, the β-elimination reaction, the reaction mode (endo/exo type), and the cleavage site (main/side chain type). In this study, the three-dimensional structures of xanthan lyase and its complex with the product were determined by x-ray crystallography at 2.3 and 2.4 Å resolution, respectively. We also identified the active cleft of the enzyme and aa residues responsible for both the recognition of the substrate and the catalytic reaction. Pyruvylated xanthan (average molecular mass, 2 × 106; pyruvylation of the terminal mannosyl residue in the side chain, ∼50%) was obtained from Kohjin Co., Tokyo, Japan. Polyethylene glycol 4000 was purchased from Nacalai Tesque, Kyoto, Japan. DEAE-Toyopearl 650 m and Super Q-Toyopearl 650C were from Tosoh Co., Tokyo, Japan. Bio-Gel P2 was from Bio-Rad. The restriction endonucleases and DNA-modifying enzymes were from Takara Shuzo Co., Kyoto, and Toyobo Co., Tokyo, respectively. Xanthan lyase was assayed as described previously (16Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 1998; 64: 3765-3768Crossref PubMed Google Scholar). Briefly, the enzyme was incubated in 1 ml of a reaction mixture containing 0.05% xanthan and 50 mmsodium acetate buffer, pH 5.5, and then the activity was determined by monitoring the increase in absorbance at 235 nm. One unit of the enzyme activity was defined as the amount of enzyme required to produce an increase of 1.0 in absorbance at 235 nm/min. Protein was determined by the method of Lowry et al. (22Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard or by measuring the absorbance at 280 nm, assuming that E280 = 2.06 corresponded to 1 mg/ml, as calculated from the aa sequence using ProtParam (www.expasy.org/tools/protparam.html). Unless otherwise specified, all operations were carried out at 0–4 °C. Cells ofEscherichia coli strain BL21(DE3)pLysS harboring a plasmid (pET17b-XL4) (21Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 2001; 67: 713-720Crossref PubMed Scopus (23) Google Scholar) were grown in 6 liters of LB medium (1.5 liters/flask), collected by centrifugation at 6000 × gand 4 °C for 5 min, washed with 20 mm potassium phosphate buffer (KPB), pH 7.0, and then resuspended in the same buffer. The cells were disrupted ultrasonically (Insonator model 201M, Kubota, Tokyo, Japan) at 0 °C and 9 kHz for 20 min, and the clear solution obtained upon centrifugation at 15,000 × gand 4 °C for 20 min was used as the cell extract containing the precursor form (97 kDa) of the enzyme. The cell extract, after supplementation with 1 mm phenylmethylsulfonyl fluoride and 0.1 μm pepstatin A, was fractionated with ammonium sulfate. The precipitate (0–30% saturation) was collected by centrifugation at 15,000 × g and 4 °C for 20 min, dissolved in 20 mm KPB, pH 7.0, and then applied to a DEAE-Toyopearl 650 m column (2.6 × 15 cm) equilibrated with 20 mm KPB, pH 7.0. The enzyme was eluted with a linear gradient of NaCl (0–0.7 m) in 20 mm KPB, pH 7.0 (200 ml), with 2 ml fractions collected every 2 min. The active fractions, which were eluted with 0.4m NaCl, were combined and dialyzed against 20 mm KPB, pH 7.0. The dialysate was used as the purified precursor form (97 kDa) of the enzyme. To convert the precursor (97 kDa) autocatalytically to the mature form (∼75 kDa), the purified precursor was kept at 4 °C for 1 week. After confirmation of the conversion by SDS-PAGE (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), the enzyme solution was applied to a Super Q-Toyopearl 650C column (1.7 × 5 cm) equilibrated with 20 mm KPB, pH 7.0, and eluted with a linear gradient of NaCl (0 to 0.5 m) in 20 mm KPB, pH 7.0 (50 ml). 1-ml fractions were collected every minute. The active fractions, which were eluted with about 0.2 m NaCl, were combined and dialyzed against 20 mm Tris-HCl, pH 7.5, and the dialysate was used as the purified mature form (∼75 kDa) of the enzyme. Xanthan (0.5%) dissolved in 50 mm sodium acetate (pH 5.5) (200 ml) was treated with the purified xanthan lyase (10 mg). The solution was mixed with ethanol (400 ml) and then centrifuged at 15,000 × g and 4 °C for 20 min. The supernatant was concentrated to 1.5 ml through evaporation and then applied to a Bio-Gel P2 column (0.9 by 122 cm) equilibrated with distilled water. The sugar eluted was determined to be PyrMan by confirming the release of mannose and pyruvate on hydrolysis with trifluoroacetic acid, as described previously (16Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 1998; 64: 3765-3768Crossref PubMed Google Scholar). The fractions containing PyrMan were collected, freeze-dried, and dissolved in distilled water. The purity and content of PyrMan were determined by TLC analysis as described previously (16Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 1998; 64: 3765-3768Crossref PubMed Google Scholar). The mature form (∼75 kDa) of xanthan lyase was crystallized by the hanging-drop vapor diffusion method. The solution for a crystallization drop was prepared on a siliconized coverslip by mixing 3 μl of protein solution (7.18 mg of protein/ml) with 3 μl of mother liquor comprising 23% polyethylene glycol 4000, 0.2 m ammonium formate, and 0.1m sodium Bicine buffer, pH 9.0. The crystals were soaked in several heavy atom derivative solutions comprising 2 mmNaAuCl4, 0.2 mm AgNO3, 1 mm Ac2UO2, 2 mmSmCl3, 1 mm GdCl3, 1 mmHoCl3, and 1 mm CdCl2 for 1 or 2 h at 20 °C. Crystals were also soaked in a sugar solution containing 75 mm PyrMan. All heavy atom and sugar solutions were prepared with a modified mother liquor consisting of 23% polyethylene glycol 4000, 0.2 m ammonium formate, and 0.1m Tris-HCl buffer, pH 7.4. Diffraction data for the native and derivative crystals were collected with a Bruker Hi-Star multiwire area detector at 20 °C, using CuKα radiation generated by a MAC Science M18XHF rotating anode generator, and were processed with the SADIE and SAINT software packages (Bruker, Karlsruhe, Germany) (TableI).Table IData collection and refinement statistics for xanthan lyaseNativeComplexCrystal systemOrthorhombicOrthorhombicSpace groupP212121P212121Cell dimensions a, b, c (Å)54.323, 91.446, 160.74554.357, 91.305, 159.258Molecules/asymmetric unit11Data collectionX-ray sourceRotating anodeRotating anodeDetectorBruker Hi-starBruker Hi-starResolution range (last shell)aThe data in the highest resolution shells are given in parentheses.(Å)27.6–2.22 (2.30–2.22)26.3–2.40 (2.49–2.40)Measured reflections (last shell)112,089 (2,776)55,703 (4,006)Unique reflections (last shell)34,941 (1,632)28,677 (2,308)Completeness (last shell) (%)86.4 (58.0)89.9 (53.4)R-sym (last shell) (%)8.4 (36.8)9.1 (37.6)RefinementResolution (last shell) (Å)50–2.3 (2.38–2.30)50–2.4 (2.49–2.40)Used reflections (last shell)30,582 (2,098)26,294 (1,716)Completeness (last shell) (%)83.9 (58.3)82.6 (54.8)Residues/water752/248752/261Calcium ion11PyrMan1Average B-factor (Å2)28.120.0r.m.s. deviationBond length (Å)0.00600.0062Bond angle (deg)1.291.31R-factor (last shell) (%)17.5 (26.6)16.9 (26.0)R-free (last shell) (%)24.0 (34.4)24.2 (32.0)a The data in the highest resolution shells are given in parentheses. Open table in a new tab The crystal structure of xanthan lyase was solved by the multiple isomorphous replacement (m.i.r.) method. The major sites of heavy atoms were determined by interpretation of the peaks in difference Patterson maps obtained at 3.0 Å resolution. Additional heavy atom sites were determined from the peaks in difference Fourier maps. Phase refinement was performed with the program package PHASES (24Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar). The results of heavy atom refinement and phasing by m.i.r. at 3.0 Å resolution are presented in Table II. The phase was improved greatly and the figure-of-merit increased to 0.854 after solvent flattening with PHASES (25Wang B.C. Methods Enzymol. 1985; 115: 90-112Crossref PubMed Scopus (939) Google Scholar). The model was built using the program TURBO-FRODO (AFMB-CNRS, Marseille, France) on a Silicon Graphics Octane computer. Simulated annealing refinement was carried out with this model using 50–2.3 Å resolution data obtained with CNS (26Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). The model was heated to 2500 K and then slowly cooled to 300 K (time-step, 0.5 fs; decrease in temperature, 25 K; number of steps at each temperature, 50), and then 150 cycles of Powell minimization were carried out. Fo − Fc and 2 Fo − Fc maps were used to locate the correct model. Several rounds of positional andB-factor refinement followed by manual model building were carried out to improve the model by increasing the data to 2.3 Å resolution. Water molecules were incorporated where the difference in density was more than 3.0 ς above the mean and the 2 Fo − Fc map showed a density of more than 1.0 ς. The final R-factor was 17.5% for 30,582 data points in the 50.0–2.3 Å resolution range (83.9% completeness). The R-free value calculated for the randomly separated 10% data was 24.0%.Table IIStatistics for heavy atom derivatives of xanthan lyaseDerivativeConditionsaThe soaking solution comprised 23% polyethylene glycol 4000, 0.2 m ammonium formate, and 0.1 mTris-HCl, pH 7.4.Res. (Å)R CullisR KrautPhasing powerHeavy atomSiteOccupancyBXYZmm, hÅ2NaAuCl42, 24.00.7530.0860.52Au-10.8260.1730.1800.55049.2AgNO30.2, 24.00.7890.0600.38Ag-10.2480.5210.0420.53767.0UO2Ac21, 13.00.6100.1451.17U-10.9630.9590.6841.1552.0U-20.5370.9590.6971.40261.0SmCl32, 13.00.6770.0951.43Sm-10.1190.0600.5810.80071.8Sm-20.9610.9590.6831.59010.4Sm-30.5430.9560.6970.38047.0GdCl31, 13.00.6710.0851.47Gd-10.5390.0410.1841.71612.1Gd-20.9560.0430.1970.19333.1Gd-30.1180.0610.5810.83865.1HoCl31, 13.00.6740.0831.53Ho-10.5380.0410.1841.3232.0Ho-20.1190.0600.5810.47436.8CdCl21, 13.00.7310.0841.31Cd-10.5360.0420.1841.6742.0Cd-20.9630.0410.2000.25312.4a The soaking solution comprised 23% polyethylene glycol 4000, 0.2 m ammonium formate, and 0.1 mTris-HCl, pH 7.4. Open table in a new tab A crystal soaked with PyrMan was isomorphous with the crystal used for the native set. A Fo − Fc map (contoured at 3.0 ς) at 2.4 Å resolution was obtained using the reflection data for the sugar-soaked crystal, and the phase was calculated from the final model of xanthan lyase. The stereo quality of the model was assessed using the programs PROCHECK (27Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHAT-CHECK (28Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1788) Google Scholar). Ribbon plots were prepared using the programs MOLSCRIPT (29Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BOBSCRIPT (30Esnouf R.M. J. Mol. Graph. Model. 1997; 15 (112–113): 132-134Crossref PubMed Scopus (1794) Google Scholar), RASTER3D (31Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar), and GRASP (32Nicholls A. Sharp K. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Crossref PubMed Scopus (5311) Google Scholar). The coordinates of lyases for hyaluronate and chondroitin AC were taken from the RCSB Protein Data Bank (33Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (26227) Google Scholar). These molecular models were superimposed by means of fitting the programs RIGID and TOP included in TURBO-FRODO and CCP4, respectively. The mature form (about 75 kDa) of xanthan lyase of Bacillus sp. GL1 was purified from recombinant E. coli cells harboring plasmid pET17b-XL4 (21Hashimoto W. Miki H. Tsuchiya N. Nankai H. Murata K. Appl. Environ. Microbiol. 2001; 67: 713-720Crossref PubMed Scopus (23) Google Scholar). A crystal of xanthan lyase (0.3 × 0.2 × 0.05 mm) was obtained by the hanging-drop vapor diffusion method. The space group was determined to beP212121 (orthorhombic) with unit cell dimensions of a = 54.3 Å,b = 91.4 Å, and c = 160.7 Å; the solvent content was 50.2% assuming one molecule/asymmetric unit. The results of the x-ray data collection are summarized in TableI. The structure of the enzyme was determined by the m.i.r. method. Table IIshows the refinement statistics for the heavy atoms at 3.0 Å resolution. The protein model was built after solvent flattening of the m.i.r. phase with the PHASES program (24Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar), and the model was refined by means of simulated annealing and the restrained least-squares method using CNS (26Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar), as shown in Table I. The refined model of xanthan lyase comprises 752 aa residues, 248 water molecules, and one calcium ion. The N- and C-terminal aa residues of the mature form produced from the preproform through posttranslational processing were confirmed to be Ser26 and Gly777, respectively, by electron density mapping. All of the polypeptide chain sequences could be well traced, and the electron density of the main and side chains was generally very well defined in the 2 Fo − Fc map. The final overallR-factor for the refined model was 0.175, with 30,582 unique reflections within the 50.0–2.3 Å resolution range. The final freeR-factor calculated with the randomly selected 10% data was 0.240. The final root-mean-square (r.m.s.) deviations from the standard geometry were 0.0060 Å for bond lengths and 1.29o for bond angles. Based on the theoretical curves in the plot calculated according to Luzzati (34Luzzati V. Acta Crystallogr. 1952; 5: 802-810Crossref Google Scholar), the absolute positional error was estimated to be close to 0.25 Å of 5.0–2.3 Å resolution. Judging from the results of Ramachandran plot analysis, in which the stereochemical correctness of the backbone structure is indicated by the (φ, ψ) torsion angles (35Ramachandran G.N. Sasisekharan V. Adv. Protein Chem. 1968; 23: 283-437Crossref PubMed Scopus (2736) Google Scholar), most of the non-glycine residues (86.1%) lay within the most favored regions, and the other residues (13.6%) fell in the additional and generously allowed regions, except for the Thr247 and Asp695 residues. The Thr247 and Asp695 residues are in β-turns. In particular, the latter turn, containing the Asp695 residue, is similar to a β-hairpin consisting of four amino acid residues with the conformation of β-ε-γ-β in a Ramachandran plot (36Sibanda B.L. Thornton J.M. Nature. 1985; 316: 170-174Crossref PubMed Scopus (507) Google Scholar), because the Ala694, Asp695, Leu696, and Ile697 residues fell in or near the β, ε, γ, and β regions of the plot, respectively. Furthermore, there is one cis-peptide between the Ala753 and Pro754residues. Figs.2 and 3 depict a ribbon model of the overall structure and topology of the secondary structure elements of xanthan lyase, respectively. The enzyme has approximate dimensions of 100 × 70 × 50 Å and is composed of two globular domains (N- and C-terminal domains) that form α- and β-structures, respectively. The N-terminal domain comprises the 352 aa residues from Ser26 to Asp377 and is composed predominantly of 13 α-helices, 10 of which form an α/α barrel structure. The C-terminal domain comprises the 389 aa residues from Leu389 to Gly777 and one calcium ion and consists of 30 β-strands arranged in five anti-parallel β-sheets. A peptide linker composed of the 11 aa residues from Asp378to Asn388 connects the N- and C-terminal domains. In the structure of xanthan lyase, 25.7% of all aa residues are in α-helices, 26.2% in β-strands, and the remaining 48.1% in turns and coils.Figure 3Topology diagram of xanthan lyase. The enzyme consists of two domains (N- and C-terminal domains). The 13 α-helices (HA1–HA13) and two short β-strands (S1 and S2) in the N-terminal domain are shown asboxes and arrows, respectively. The inner helices (HA3, HA5, HA7, HA9, andHA11) and outer helices (HA4, HA6,HA8, HA10, and HA12) in the α/α-barrel structure are shown in green andpurple, respectively, and the other helices (HA1,HA2, and HA13) are in gray. The 30 β-strands arranged into sheets A, B,C, D, and E in the C-terminal domain are shown as blue, purple, cyan, green, and red arrows, respectively. A short α-helix (HB1) and calcium ion (Ca) are shown as a gray box and ayellow ball, respectively.View Large Image Figure ViewerDownload (PPT) The N-terminal domain is composed primarily of two short β-strands and 13 α-helices. The latter contribute to the formation of an α/α-barrel structure with a deep cleft, which is considered to be an active site (Figs. 2 and4 A). The 13 major α-helices, numbered sequentially from HA1 to HA13 (HA1, aa residues 28–41; HA2, 51–69; HA3, 90–106; HA4, 118–131; HA5, 153–165; HA6, 173–184; HA7, 192–208; HA8, 212–221; HA9, 255–270; HA10, 284–291; HA11, 318–333; HA12, 338–354; and HA13, 366–376), vary in length between 8 and 19 aa residues and consist of 187 aa residues (Fig. 3). These α-helices are arranged as follows: four (HA5, HA6, HA8, and HA13) with three turns; seven (HA1, HA3, HA4, HA7, HA9, HA11, and HA12) with four turns; one (HA2) with five turns; and one (HA10) with two turns. There are 12 loops (from LA1 to LA12) connecting an α-helix to the following α-helix in the N-terminal domain (Fig. 3). The loop (LA8) between HA8 and HA9 includes two short β-strands (S1, aa residues 235–238, and S2, 243–245). Therefore, in the N-terminal domain, 52.3% of all aa residues are in α-helices, 2.0% in β-strands, and the remaining 45.7% in turns and coils. The N-terminal α-helical domain includes an incomplete α5/α5-barrel formed by five inner and five outer α-helices, and the 10 α-helices (from HA3 to HA12) constituting the α5/α5-barrel are located within the core of the domain (Fig. 4 A). These 10 helices are connected by short and long loops in a nearest neighbor, up-and-down pattern. This arrangement is described as a “twisted α/α-barrel” with five inner α-helices (HA3, HA5, HA7, HA9, and HA11), which are oriented in roughly the same direction, and five outer α-helices (HA4, HA6, HA8, HA10, and HA12) running in the opposite direction. The C-terminal domain consists predominantly of 30 β-strands (SA1, aa residues 389–396; SA2, 398–402; SA3, 407–" @default.
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• W1966354566 title "Crystal Structure of Bacillus sp. GL1 Xanthan Lyase, Which Acts on the Side Chains of Xanthan" @default.
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