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• W2048510003 abstract "Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 Å resolution with a final R-factor of 16.8% for 25 to 1.8 Å resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an α6/α6-barrel, whose structure is found in the six-hairpin enzyme superfamily of an α/α-toroidal fold. One side of the UGL α6/α6-barrel structure consists of long loops containing three short β-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp88 and Asp149. This was supported by site-directed mutagenesis studies in Asp88 and Asp149. Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 Å resolution with a final R-factor of 16.8% for 25 to 1.8 Å resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an α6/α6-barrel, whose structure is found in the six-hairpin enzyme superfamily of an α/α-toroidal fold. One side of the UGL α6/α6-barrel structure consists of long loops containing three short β-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp88 and Asp149. This was supported by site-directed mutagenesis studies in Asp88 and Asp149. Polysaccharides exist ubiquitously in nature as components of the extracellular matrix on the cell surface of many different organisms, ranging from bacteria to mammals (1Scott J.E. J. Anat. 1995; 187: 259-269Google Scholar). These polysaccharides are important to a variety of biological and functional activities, and are divided into three groups: i.e. storage, e.g. starch; structural, e.g. cellulose; and functional, e.g. glycosaminoglycan. Glycosaminoglycans such as hyaluronan, chondroitin, and heparin are linear, negatively charged polysaccharides with a repeating disaccharide unit consisting of a uronic acid residue (glucuronic or iduronic acid) and an amino sugar residue (glucosamine or galactosamine) (2Ernst S. Langer R. Cooney C.L. Sasisekharan R. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 387-444Google Scholar). Hyaluronan consists of d-glucuronic acid (GlcA) 1The abbreviations used are: GlcA, d-glucuronic acid; UGL, unsaturated glucuronyl hydrolase; DTT, dithiothreitol; MPD, 2-methyl-2,4-pentanediol; GlcNAc, N-acetyl-d-glucosamine; ΔGlcA, unsaturated d-glucuronic acid; GalNAc, N-acetyl-d-galactosamine; Glc, d-glucose; Rha, l-rhamnose; Man, d-mannose; mir, multiple isomorphous replacement; r.m.s., root mean square; AGE, N-acyl-d-glucosamine 2-epimerase. and N-acetyl-d-glucosamine (GlcNAc) (Fig. 1a) and plays an important role in cell-to-cell association in mammals and as a capsule in streptococcal bacteria (3Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Google Scholar). This polysaccharide is widely present in such human tissues as the eye, brain, liver, skin, and blood (4Lindahl U. Hook M. Annu. Rev. Biochem. 1978; 47: 385-417Google Scholar). Chondroitin, also a member of the glycosaminoglycan family, consists of GlcA and N-acetyl-d-galactosamine (GalNAc) with a sulfate group(s) at position 4 or 6 or both (5Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Google Scholar) (Fig. 1b). Mammalian chondroitin covalently bound with proteins plays an important role in cellular architecture and permeability (5Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Google Scholar). These glycosaminoglycans in the extracellular matrix may be a target for pathogens that invade host cells, and many pathogens have been reported to show specific interaction with these polysaccharides (6Sawitzky D. Med. Microbiol. Immunol. (Berl.). 1996; 184: 155-161Google Scholar). Certain streptococci such as Streptococcus pyogenes and Streptococcus pneumoniae cause severe infectious disease, e.g. pneumonia, bacteremia, sinusitis, and meningitis, and produce polysaccharide lyases, which function as virulence factors in the degradation of hyaluronan and chondroitin (7Linker A. Meyer K. Weissmann B. J. Biol. Chem. 1955; 213: 237-248Google Scholar, 8Boulnois G.J. J. Gen. Microbiol. 1992; 138: 249-259Google Scholar). Lyases for haluronan and chondroitin recognize GlcA residues in polysaccharides and produce unsaturated disaccharides with a GlcA residue having a C=C double bond at the nonreducing terminus through the β-elimination reaction. Unsaturated glucuronyl hydrolase (UGL) catalyzes the hydrolysis of unsaturated disaccharides to an amino sugar and an unsaturated GlcA (ΔGlcA), which is nonenzymatically converted immediately to α-keto acid (Fig. 1) (9Hashimoto W. Kobayashi E. Nankai H. Sato N. Miya T. Kawai S. Murata K. Arch. Biochem. Biophys. 1999; 368: 367-374Google Scholar). The enzyme is thus thought to be another virulent factor responsible for the complete degradation of glycosaminoglycans. Although a gene coding for UGL was first cloned from Bacillus sp. GL1 that degrades bacterial biofilms such as xanthan, which is produced by pathogenic xanthomonads, and gellan, which is produced by pathogenic sphingomonads (Fig. 1, c and d) (10Hashimoto W. Maesaka K. Sato N. Kimura S. Yamamoto K. Kumagai H. Murata K. Arch. Biochem. Biophys. 1997; 339: 17-23Google Scholar, 11Nankai H. Hashimoto W. Miki H. Kawai S. Murata K. Appl. Environ. Microbiol. 1999; 65: 2520-2526Google Scholar), highly homologous genes are distributed in pathogenic streptococci that produce polysaccharide lyases (12Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Google Scholar). We thus defined UGL as a novel member of hydrolase for the degradation of glycosaminoglycans and bacterial biofilms, and the enzyme belongs to a new glycoside hydrolase family, GH-88, in the CAZY data base. 2B. Henrissat, P. Coutinho, and E. Deleury, afmb.cnrs-mrs.fr/~cazy/CAZY/index.html. Inhibitors of polysaccharide lyases and UGL are expected to become potent pharmaceuticals for treating streptococci infectious disease. Structural analysis of polysaccharide lyases and UGL is indispensable for clarifying molecular mechanisms underlying catalysis and recognition of substrates, and sequential reaction mechanisms involved in polysaccharide depolymerization by bacteria. The crystal structures of polysaccharide lyases such as those for pectate (13Yoder M.D. Keen N.T. Jurnak F. Science. 1993; 260: 1503-1507Google Scholar, 14Akita M. Suzuki A. Kobayashi T. Ito S. Yamane T. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2001; 57: 1786-1792Google Scholar, 15Charnock S.J. Brown I.E. Turkenburg J.P. Black G.W. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 99: 12067-12072Google Scholar), alginate (16Yoon H.-J. Mikami B. Hashimoto W. Murata K. J. Mol. Biol. 1999; 290: 505-514Google Scholar), hyaluronate (17Li S. Kelly S.J. Lamani E. Ferraroni M. Jedrzejas M.J. EMBO J. 2000; 19: 1228-1240Google Scholar), chondroitin (18Jansson P.E. Lindberg B. Sandford P.A. Carbohydr. Res. 1983; 124: 135-139Google Scholar, 19Féthière J. Eggimann B. Cygler M. J. Mol. Biol. 1999; 288: 635-647Google Scholar), and xanthan (20Hashimoto W. Nankai H. Mikami B. Murata K. J. Biol. Chem. 2003; 278: 7663-7673Google Scholar) have been determined, and the structural and functional relationship of these lyases have been studied. No three-dimensional structure has been clarified for any UGL, however. This article deals with the crystal structure of UGL, a novel hydrolase for the degradation of glycosaminoglycans and bacterial biofilms, from Bacillus sp. GL1 determined by x-ray crystallography at 1.8 Å resolution and the identification of the active site. The structure provides useful information on the catalytic mechanism and for the molecular design of drugs for the treatment of streptococci, xanthomonad, and sphingomonad infectious diseases. Crystallization and X-ray Diffraction—UGL of Bacillus sp. GL1 was overexpressed in Escherichia coli, purified, and crystallized by sittingdrop vapor diffusion as described elsewhere (21Mori S. Akao S. Miyake O. Nankai H. Hashimoto W. Mikami B. Murata K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 946-949Google Scholar). UGL crystals were soaked in a heavy atom derivative solution containing 1 mm of K2PtCl4, 1 mm of Hg(CH3COO)2, or 0.5 mm of NaAuCl4 for 15–50 min at 20 °C. These heavy atom solutions were prepared in 52% (v/v) 2-methyl-2,4-pentanediol (MPD), 0.12 m of sodium chloride, 0.1 m of glycine, and 0.1 m of Tris-HCl buffer (pH 7.6). The crystals we used were removed from a droplet on a mounted nylon loop (Hampton Research, Laguna Niguel, CA), and placed in a cold nitrogen gas stream at 100 K. X-ray diffraction images of the UGL crystal (Native 1) were collected using a Quantum 4R CCD area detector (ADSC) with synchrotron radiation at a wavelength of 0.72 Å at the BL-38B1 station of SPring-8. Images were processed with DENZO and SCALEPACK software (22Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar) to a resolution of 1.8 Å (Table I). Diffraction images of another crystal (Native 2) and derivative crystals for phasing were collected with a Bruker Hi-Star multiwire area detector using CuKα radiation generated by a MAC Science M18XHF rotating anode generator, and were processed with SADIE and SAINT software (Bruker, Karlsruhe, Germany).Table ISynchrotron radiation data collection and refinement statistics for a UGL crystal (Native 1)Crystal systemHexagonalSpace groupP6522Unit cell parameters (Å)a = b = 103.01, c = 223.04Molecules/asym. unit1Data collectionResolution limit (last shell)aData in highest resolution shells are given in parentheses (Å)25.0–1.80 (1.86–1.80)Measured reflections (last shell)300,292 (24083)Unique reflections (last shell)63,892 (6175)Redundancy (last shell)4.7 (3.9)Completeness (I > σI) (last shell) (%)97.6 (96.0)Rmerge (%)bRmerge = Σ|Ii – 〈I〉 /ΣIi × 100, where Ii is the intensity of individual reflection and 〈I〉 the mean intensity of all reflections9.6 (29.5)RefinementFinal model377 residues, 478 water, 4 glycine, 2 DTT, and 1 MPDResolution limit (last shell) (Å)25.0–1.80 (1.91 -1.80)Used reflections (last shell)63,316 (9024)Completeness (F > 2σ F) (last shell) (%)96.7 (93.9)Average B-factor (Å2)16.3R-factor (last shell) (%)cR-factor = ΣFo – Fc/Σ|Fo| × 100, where Fo is the observed structure factor and Fc the calculated structure factor16.8 (22.4)Rfree (last shell) (%)dRfree was calculated from a randomly chosen 10% of reflections as defined by the CNS18.9 (24.6)R.m.s. deviationsBond (Å)0.005Angle (deg)1.22a Data in highest resolution shells are given in parenthesesb Rmerge = Σ|Ii – 〈I〉 /ΣIi × 100, where Ii is the intensity of individual reflection and 〈I〉 the mean intensity of all reflectionsc R-factor = ΣFo – Fc/Σ|Fo| × 100, where Fo is the observed structure factor and Fc the calculated structure factord Rfree was calculated from a randomly chosen 10% of reflections as defined by the CNS Open table in a new tab Structure Determination and Refinement—The UGL crystal structure was determined by multiple isomorphous replacement (mir). Phase calculation and refinement were done with Native 2 and derivative data sets using a PHASES program (23Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Google Scholar). Major sites of heavy atoms were determined by the interpretation of difference Patterson maps calculated at a resolution of 6.0–3.5 Å. Additional heavy atom sites were determined from difference Fourier maps. Phasing results are listed in Table II. The mean figure of merit was 0.426. The phase was greatly improved and the mean figure of merit increased to 0.754 after solvent flattening (24Wang B.C. Methods Enzymol. 1985; 115: 90-112Google Scholar) with a PHASES program. Initial model building was done with Native 2 data sets and the phase at 3.5 Å using the TURBO-FRODO program (AFMB-CNRS, Marseille, France) on a Silicon Graphics Octane computer. Simulated annealing refinement was done with this model and 25–2.5 Å resolution data from Native 1 data sets with CNS ver. 1.1 (25Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Google Scholar). The model was heated to 3,000 K, then slowly cooled to 300 K (time step, 0.5 fs; decrease in temperature, 25 K; number of steps at each temperature, 50), and 200 cycles of Powell minimization were done. Fo - Fc and 2 Fo - Fc maps were used to locate the correct model. Several rounds of positional and B-factor refinement, followed by manual model building, were done to improve the model by increasing data to a resolution of 1.8 Å. Water molecules were incorporated where the difference density exceeded the mean by 3.0 σ or more and the 2 Fo - Fc map showed a density exceeding 1.0 σ. Seven fragments of nonprotein or nonwater density were modeled into four glycine molecules, two dithiothreitol (DTT) molecules, and one MPD molecule from the crystallization medium, and density was excellent for the whole molecule. The final R-factor was 16.8% for 63,316 data points with F > 2.0σ (F) in a resolution of 25.0–1.8 Å (96.7% completeness). The R-free value calculated for randomly separated 10% data was 18.9%. The stereoquality of the model was assessed using the PRO-CHECK (26Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallog. 1993; 26: 283-291Google Scholar) and WHAT-CHECK (27Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Google Scholar) programs. Structural similarity was searched for in the RCSB Protein Data Bank (28Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Google Scholar) using the DALI program (29Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Google Scholar). Coordinates of hypothetical protein Yter (1NC5) were taken from the RCSB Protein Data Bank. UGL and Yter models were superimposed by a fitting program in TURBO-FRODO. Ribbon plots were prepared using the MOLSCRIPT (30Kraulis P.J. J. Appl. Crystallog. 1991; 24: 946-950Google Scholar), RASTER3D (31Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1994; 50: 869-873Google Scholar), and GRASP (32Nicholls A. Sharp K. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Google Scholar) programs.Table IIPhasing statisticsCompoundNative 2K2PtCl4Hg(CH3COO)2NaAuCl4Concentration (mm)aSoaking solution consisted of 52% (v/v) MPD, 0.12 m of sodium chloride, 0.1 m of glycine, and 0.1 m of Tris-HCl buffer (pH 7.6)1.01.00.5Soaking time (min)503015Resolution limit (Å)2.73.53.53.5Phasing power1.081.231.06Rcullis0.6120.6520.701Rkraut0.0880.2110.104Number of sites222Binding sitesMet1, Met121Cys150, Cys287Cys150, His210Mean figure of merit0.426a Soaking solution consisted of 52% (v/v) MPD, 0.12 m of sodium chloride, 0.1 m of glycine, and 0.1 m of Tris-HCl buffer (pH 7.6) Open table in a new tab Mutagenesis and CD Spectra Measurement—Asp88 or Asp149 in UGL was replaced with an asparagine residue by the use of the QuikChange site-directed mutagenesis kit (Stratagene), and mutation was confirmed by DNA sequencing. The plasmid pET3a-UGL, which is the expression vector for wild-type UGL (12Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Google Scholar), was used as a template. Primers were as follows: D88N (Asp88 → Asn88), 5′-AGAATCTCGATCATCACAACATCGGCTTCCTATACTC-3′ and 5′-GAGTATAGGAAGCCGATGTTGTGATGATCGAGATTCT-3′; and D149N (Asp149 → Asn149), 5′-GGACGCATCATCATCAACTGCCTGCTGAATCTG-3′ and 5′-CAGATTCAGCAGGCAGTTGATGATGATGCGTCC-3′ (mutations are indicated by bold letters). The E. coli BL21 (DE3) was transformed with plasmids having a mutation, i.e. pET3a-UGL(D88N) and pET3a-UGL(D149N). Mutant enzymes were expressed and purified as for wild-type UGL (12Mori S. Akao S. Nankai H. Hashimoto W. Mikami B. Murata K. Protein Expr. Purif. 2003; 29: 77-84Google Scholar). Structural conformations of purified wild-type and mutant enzymes were evaluated by far-UV CD spectroscopy using a Jasco J720 spectropolarimeter at 190–260 nm with a demountable quartz cell having a 0.1-mm path length. Enzyme Assay—UGL reactions for the wild-type and mutants were conducted at 30 °C as follows: The reaction mixture consisted of 50 mm of sodium phosphate buffer (pH 6.5), 20–500 μm of substrate, and enzymes in a 500-μl reaction volume. Enzyme activity was measured by monitoring the decrease in absorbance at 235 nm, corresponding to the loss of the C=C double bond of the substrate because the pyranose ring of the released ΔGlcA readily opens so that it is nonenzymatically converted to α-keto acid through the loss of the double bond (Fig. 1d) (7Linker A. Meyer K. Weissmann B. J. Biol. Chem. 1955; 213: 237-248Google Scholar, 9Hashimoto W. Kobayashi E. Nankai H. Sato N. Miya T. Kawai S. Murata K. Arch. Biochem. Biophys. 1999; 368: 367-374Google Scholar). Enzyme concentration was determined by UV spectrophotometry using theoretical molar extinction coefficient ϵ280 = 99,570 (m-1 cm-1). Enzyme purity was assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining. The gellan lyase product (ΔGlcA-Glc-Rha-Glc) for the UGL substrate was prepared as described elsewhere (10Hashimoto W. Maesaka K. Sato N. Kimura S. Yamamoto K. Kumagai H. Murata K. Arch. Biochem. Biophys. 1997; 339: 17-23Google Scholar). Hyaloronate lyase products (ΔGlcA-GlcNAc) and chondroitin lyase products (ΔGlcA-GalNAc) were obtained from Seikagaku Corporation (Tokyo, Japan). kcat and Km parameters were determined by nonlinear fitting to the Michaelis-Menten equation. Crystallization and Structure Determination—UGL of Bacillus sp. GL1 is a monomeric enzyme with a molecular mass of about 43 kDa (377 amino acid residues) (9Hashimoto W. Kobayashi E. Nankai H. Sato N. Miya T. Kawai S. Murata K. Arch. Biochem. Biophys. 1999; 368: 367-374Google Scholar). A UGL crystal (0.1 × 0.1 × 0.5 mm) was obtained by sitting-drop vapor diffusion as described elsewhere (21Mori S. Akao S. Miyake O. Nankai H. Hashimoto W. Mikami B. Murata K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 946-949Google Scholar). The space group was determined to be P6522 with unit cell dimensions of a = b = 103.01 and c = 223.04 Å, and the solvent content was 69% assuming one molecule per asymmetric unit. Results of native data collection using synchrotron radiation at the BL-38B1 station of SPring-8 are summarized in Table I. The phase of the structure was solved by mir. Table II shows phasing statistics at a resolution of 3.5 Å. The protein model was built after solvent flattening with the PHASES program (23Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Google Scholar) and refined by simulated annealing and the restrained least-squares method using CNS (25Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Google Scholar) (Table I). Quality of Refined Model—The refined model of UGL consists of 377 amino acid residues, and 478 water molecules, four glycine molecules, two DTT molecules, and one MPD molecule. The entire polypeptide chain sequence was well traced, and the electron densities of the main chain and side chain were generally very well defined in the 2 Fo - Fc map, except for N-terminal amino acid residue Met1 and C-terminal amino acid residue Arg377, whose electron density was too low for them to be identified completely. Other ligand molecules were also well fitted. The final overall R-factor for the refined model was 16.8%, with 63,316 unique reflections at a resolution of 25.0–1.8 Å. The final free R-factor was 18.9%. Final root mean square (r.m.s.) deviations from standard geometry were 0.005 Å for bond lengths and 1.22° for bond angles. Based on theoretical curves in the plot calculated according to Luzzati (33Luzzati V. Acta Crystallogr. 1952; 5: 802-810Google Scholar), the absolute positional error was estimated to be close to 0.17 Å at a resolution of 5.0–1.8 Å. Most (88.3%) nonglycine residues lie within most favored regions, and other residues (11.3%) within additionally allowed regions of the Ramachandran plot as defined in PROCHECK (26Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallog. 1993; 26: 283-291Google Scholar). Ser345 (ϕ = 38°, Ψ = 65°), however, falls in generously allowed regions, exhibiting well defined density in the 2 Fo - Fc map and located in a sharp bend of the loop neighboring a helix. There are no residues in disallowed regions. Overall UGL Structure—The overall structure of UGL is shown as ribbon models (Fig. 2, a and b) and a molecular surface model (Fig. 2c). The enzyme is ∼45 × 45 × 40 Å and consists of an α6/α6-barrel structure with a deep pocket that is likely to be the active site (described below). Fig. 3 shows the UGL structure topology. The structure consists of 12 long α-helices (H1, 3–20; H2, 44–58; H3, 61–79; H4a, 89–94; H4b, 96–104; H5, 107–120; H6, 154–164; H7, 167–183; H8, 220–237; H9, 240–254; H10, 279–294; H11, 301–320; and H12, 351–366), 3 antiparallel β-sheets consisting of 2–3 strands and designated SA to SC (SA1, 24–28; SA2, 34–37; SB1, 124–125; SB2, 130–131; SC1, 145–147; SC2, 194–197; and SC3, 204–208), 2 short 310-helices (121–123 and 148–153) adjoining the long α-helices, and some loops. H4 has one additional residue, Leu95, whose oxygen atom has no hydrogen bond with the nitrogen atom of a paired residue. This α-helix (H4) was thus divided into two segments, H4a and H4b. The α-helix bends at this point, the angle being 30° between H4a and H4b. The α6/α6-barrel structure is formed by six outer helices (H1, H3, H5, H7, H9, and H11) running in roughly the same direction and six inner helices (H2, H4, H6, H8, H10, and H12) oriented in the opposite direction. These helices are connected, in a nearest neighbor and an up and down pattern by short and long loops. Loops between α-helices, such as H1 and H2, are referred to as L-H1:H2. One side of the barrel has short loops (L-H2:H3, L-H4:H5, L-H6:H7, L-H8:H9, 2 residues; and L-H10:H11, 6 residues). The other side, including the pocket, consists of long loops (L-H1:H2, 23 residues; L-H3:H4, 9 residues; L-H5:H6, 33 residues; L-H7:H8, 36 residues; L-H9:H10, 24 residues; and L-H11:H12, 30 residues). These long loops are packed together and form the wall of the deep funnel-shaped pocket roughly 15–20 Å in diameter at the lip and about 15 Å deep (Fig. 2c). This pocket is widely surrounded by aromatic residues.Fig. 3Topology of the fold in UGL. UGL includes an α6/α6-barrel. The 12 α-helices in the α6/α6-barrel are numbered from the N-terminal, H1 to H12, as in Fig. 2. Six boxes (H1, H3, H5, H7, H9, and H11) are outer α-helices facing the solvent. The other six boxes (H2, H4, H6, H8, H10, and H12) are inner α-helices. The 310-helix is also shown as boxes. The three short, antiparallel β-sheets, designated SA to SC, are shown as arrows.View Large Image Figure ViewerDownload (PPT) Structural Comparison—UGL consists of an α/α-toroidal fold. This basic fold is common in glycosyl hydrolases, polysaccharide lyases, and terpenoid cylases/protein prenyltransferases in the SCOP data base (scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.b.bcj.html) (34Murzin A.Z. Brenner S.E. Hubbard T.J. Chothia C. J. Mol. Biol. 1995; 247: 536-540Google Scholar). UGL has the α6/α6-barrel found in the six-hairpin enzyme superfamily of the SCOP data base, which includes glucoamylases (35Aleshin E.A. Golubev A. Firsov L.M. Honzatko R.B. J. Biol. Chem. 1992; 267: 19291-19298Google Scholar, 36Aleshin A.E. Feng P.-H. Honzatko R.B. Reilly P.J. J. Mol. Biol. 2003; 327: 61-73Google Scholar, 37Ševčík J. Solovicová A. Hostinová E. Gašperík J. Wilson K.S. Dauter Z. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 854-866Google Scholar), cellulase catalytic domains (38Juy M. Amit A.G. Alzari P.M. Poljak R.J. Claeyssens M. Bguin P. Aubert J. Nature. 1992; 357: 89-91Google Scholar, 39Alzari P.M. Souchon H. Dominguez R. Structure. 1996; 4: 265-275Google Scholar, 40Sakon J. Irwin D. Wilson D.B. Karplus P.A. Nat. Struct. Biol. 1997; 4: 810-818Google Scholar), N-acyl-d-glucosamine 2-epimerase (AGE) (41Itoh T. Mikami B. Maru I. Ohta Y. Hashimoto W. Murata K. J. Mol. Biol. 2000; 303: 733-744Google Scholar), the maltose phosphorylase central domain (42Egloff M.P. Uppenberg J. Haalck L. Van Tilbeurgh H. Structure. 2001; 9: 689-697Google Scholar), and hypothetical protein Yter. Based on the structural similarity in the RCSB Protein Data Bank (28Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Google Scholar) observed with the DALI (29Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Google Scholar) program, three proteins, i.e. hypothetical protein Yter from B. subtilis, glucoamylase from Thermoanaerobacterium thermosaccharolyticum (36Aleshin A.E. Feng P.-H. Honzatko R.B. Reilly P.J. J. Mol. Biol. 2003; 327: 61-73Google Scholar), and AGE from the porcine kidney (41Itoh T. Mikami B. Maru I. Ohta Y. Hashimoto W. Murata K. J. Mol. Biol. 2000; 303: 733-744Google Scholar), in the SCOP data base superfamily exhibited the highest degree of similarity. These proteins exhibited Z-scores of 32.7, 21.1, and 19.4. The r.m.s. distance was 2.80 Å for the superim-positioning of 323 Cα atoms of UGL on those of Yter, 3.50 Å for that of 287 Cα atoms of UGL on those of glucoamylase, and 3.30 Å for that of 278 Cα atoms of UGL on those of AGE, although they exhibit no amino acid sequence similarity and catalyze different types of reactions. UGL is an exo-hydrolase acting on unsaturated oligosaccharides produced by polysaccharide lyases, while Yter is a hypothetical protein of unknown function. The crystal structure of Yter determined by the Midwest Center for Structural Genomics has not, to the best of our knowledge, been published, although its coordinates are available in the RCSB Protein Data Bank. Glucoamylase is a polysaccharide exo-hydrolase found in some prokaryotic and many eukaryotic microorganisms. AGE has the α6/α6-barrel structure we determined, and catalyzes the epimeric reaction for N-acetyl-d-glucosamine and N-acetyl-d-mannosamine (41Itoh T. Mikami B. Maru I. Ohta Y. Hashimoto W. Murata K. J. Mol. Biol. 2000; 303: 733-744Google Scholar). Fig. 4 shows the superimpositioning of UGL on Yter; their structures show the best fit overall. The 12 helices of the α6/α6-barrel are very similar for UGL and Yter in position, direction, and angle, but significant differences exist between UGL and Yter in the loop structure. The L-H1:H2 loop of UGL is longer (23 residues) than that of Yter (13 residues). The L-H7:H8 loop of UGL protrudes from the wall to the outside, while that of Yter is directed into the pocket. L-H11:H12 of UGL makes the pocket open wide, while that of Yter makes the pocket closed. Ligands in the Active Site—The UGL structure contains four glycine molecules (Gly401, 402, 403, 404), two DTT molecules (DTT501, 502), and one MPD molecule (MPD601) (Fig. 2). These molecules were present in the crystallization medium (21Mori S. Akao S. Miyake O. Nankai H. Hashimoto W. Mikami B. Murata K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 946-949Google Scholar). Glycine molecules are bound on the surface mainly through ionic interactions. The positive-charged side chains of Arg221, Arg334, and Arg303 face the carboxyl groups of three Gly401, Gly402, and Gly404 molecules, and the negative-charged side chains of Asp149 and Asp60 face amino groups of two Gly401 and Gly403 molecules. DTT and MPD molecules are bound in the cavity predominantly" @default.
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• W2048510003 title "Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution" @default.
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