Crystal Structures of Geobacillus stearothermophilus α-Glucuronidase Complexed with Its Substrate and Products
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m310098200
ISSN1083-351X
AutoresG. Golan, D. Shallom, A. Teplitsky, Galia Zaide, Smadar Shulami, Timor Baasov, V. Stojanoff, A. Thompson, Yuval Shoham, G. Shoham,
Tópico(s)Glycosylation and Glycoproteins Research
Resumoα-Glucuronidases cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the α-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the α-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp364 and Glu392, activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu285 cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu285 is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism. α-Glucuronidases cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the α-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the α-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp364 and Glu392, activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu285 cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu285 is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism. Hemicellulose and cellulose are the main components of the plant cell wall and the most abundant natural polysaccharides in nature. The natural degradation of these polymers, a key step in the carbon cycle on Earth, is carried out by microorganisms that can be found either free in the environment or in the digestive tract of higher animals. Efficient cellulolytic microorganisms typically secrete a battery of enzymes that can function either alone or as parts of larger enzymatic complexes such as the cellulosome (1.Shoham Y. Lamed R. Bayer E.A. Trends Microbiol. 1999; 7: 275-281Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 2.Shallom D. Shoham Y. Curr. Opin. Microbiol. 2003; 6: 219-228Crossref PubMed Scopus (511) Google Scholar, 3.Schwarz W.H. Appl. Microbiol. Biotechnol. 2001; 56: 634-649Crossref PubMed Scopus (523) Google Scholar). The enzymes that degrade cellulose and hemicellulose are in many cases modular and include catalytic domains of glycoside hydrolases and/or carbohydrate esterases as well as carbohydrate-binding modules that allow the soluble enzymes to adhere to insoluble substrates (4.Bourne Y. Henrissat B. Curr. Opin. Struct. Biol. 2001; 11: 593-600Crossref PubMed Scopus (360) Google Scholar). A most informative and updated classification of these and other carbohydrate active enzymes is available on the Carbohydrate-Active Enzymes (CAZy) server (available on the World Wide Web at afmb.cnrs-mrs.fr/CAZY). The glycosidic bond is one of the most stable bonds in nature, with a half-life of over 5 million years; glycosidases can accelerate the hydrolysis of these bonds by more than 1017-fold (5.Wolfenden R. Lu X. Young G. J. Am. Chem. Soc. 1998; 120: 6814-6815Crossref Scopus (205) Google Scholar). The key elements in this catalysis are the position of the catalytic residues and the distortion of the sugar ring so as to allow the stabilization of an oxocarbenium ion-like transition state (6.Zechel D.L. Withers S.G. Acc. Chem. Res. 1999; 33: 11-18Crossref Google Scholar). The enzymatic hydrolysis of glycosidic bonds results in either an overall retention or inversion of the anomeric configuration of the substrate. Retaining glycosidases follow a two-step double-displacement mechanism, usually involving two key active site carboxylic residues, one functioning as the nucleophile catalyst and the other as the acid/base catalyst. Inverting glycosidases use a single-displacement mechanism, in which usually one carboxylic acid functions as general acid and the other functions as a general base. α-Glucuronidases use the inverting mechanism to cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (MeGlcA) 1The abbreviations used are: MeGlcA, 4-O-methyl-d-glucuronic acid; GH, glycoside hydrolase family; GlcA67A, C. japonicus α-glucuronidase; AguA, G. stearothermophilus T-6 α-glucuronidase; WT, wild type; MeGlcAXyl3, 2-O-(4-O-methyl-α-d-glucuronosyl)-β-d-xylotriose; NSLS, National Synchrotron Light Source. and xylose units, which are part of short β-1,4-xylooligomers, the main products of the enzymatic hydrolysis of xylan by xylanases (7.Biely P. Vrsanska M. Tenkanen M. Kluepfel D. J. Biotechnol. 1997; 57: 151-166Crossref PubMed Scopus (492) Google Scholar, 8.Biely P. de Vries R.P. Vrsanska M. Visser J. Biochim. Biophys. Acta. 2000; 1474: 360-364Crossref PubMed Scopus (43) Google Scholar) (Fig. 1). The different bacterial and fungal α-glucuronidases are classified as glycoside hydrolase family (GH) 67, and the first crystal structure of an enzyme from this family, the α-glucuronidase from Cellvibrio japonicus (GlcA67A), was recently described (9.Nurizzo D. Nagy T. Gilbert H.J. Davies G.J. Structure. 2002; 10: 547-556Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The enzyme is folded into three domains, the central of which has a (β/α)8-barrel fold that accommodates the active site. Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) strain T-6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system (2.Shallom D. Shoham Y. Curr. Opin. Microbiol. 2003; 6: 219-228Crossref PubMed Scopus (511) Google Scholar). Many of the genes comprising this system are clustered together on the bacterial chromosome, and so far the identified components include intracellular and extracellular GH-10 xylanases; GH- 39, -43, and -52 β-xylosidases; GH-51 α-l-arabinofuranosidases; acetyl-xylan esterases; xylose catabolism genes; and transport systems (10.Gilead S. Shoham Y. Appl. Environ. Microbiol. 1995; 61: 170-174Crossref PubMed Google Scholar, 11.Bravman T. Zolotnitsky G. Belakhov V. Shoham G. Henrissat B. Baasov T. Shoham Y. Biochemistry. 2003; 42: 10528-10536Crossref PubMed Scopus (51) Google Scholar, 12.Bravman T. Mechaly A. Shulami S. Belakhov V. Baasov T. Shoham G. Shoham Y. FEBS Lett. 2001; 495: 115-119Crossref PubMed Scopus (44) Google Scholar, 13.Gat O. Lapidot A. Alchanati I. Regueros C. Shoham Y. Appl. Environ. Microbiol. 1994; 60: 1889-1896Crossref PubMed Google Scholar, 14.Shallom D. Belakhov V. Solomon D. Shoham G. Baasov T. Shoham Y. J. Biol. Chem. 2002; 277: 43667-43673Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The α-glucuronidase T-6 (AguA) is part of a 15.5-kb-long operon involved in the utilization of d-glucuronic acid (15.Shulami S. Gat O. Sonenshein A.L. Shoham Y. J. Bacteriol. 1999; 181: 3695-3704Crossref PubMed Google Scholar, 16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar). Here we present high resolution (1.5–2.0-Å) crystal structures of AguA in complex with substrates and products. These structures provide direct evidences for several mechanistic features. We suggest that the catalytic mechanism of AguA includes a conformational change of the active-site loop carrying the general acid residue Glu285, a distortion of the substrate into a conformation much closer to the flat conformation required for the oxocarbenium ion-like transition state, and charge repulsion between the acidic substrate and the general acid residue so as to allow its protonated state. The structural data, combined with biochemical analysis of catalytic mutants, suggest that the two carboxylic residues Asp364 and Glu392, serve as general bases, activating together the nucleophilic water molecule. Protein Expression and Purification—The α-glucuronidases from G. stearothermophilus strains T-6 and T-1 (AguA T-6 and AguA T-1; accession numbers AAC98128, and AAL32057, respectively) were cloned and overexpressed in Escherichia coli. The purification of the recombinant wild type and mutant enzymes was done as described before (16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar). Production of the selenomethionine derivative of AguA was carried out in the methionine auxotrophic Escherichia coli strain B834(DE3), essentially as described previously (17.Mechaly A. Teplitsky A. Belakhov V. Baasov T. Shoham G. Shoham Y. J. Biotechnol. 2000; 78: 83-86Crossref PubMed Scopus (25) Google Scholar), and protein purification proceeded as for the wild type enzyme. α-Glucuronidase activity was determined by measuring the release of MeGlcA from aldotetraouronic acid using the Milner and Avigad assay for uronic acids (18.Milner Y. Avigad G. Carbohydr. Res. 1967; 4: 359-361Crossref Scopus (184) Google Scholar), as described before (16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar). Crystallization and X-ray Diffraction Data Collection—All crystals were obtained using the hanging drop crystallization method and grown at 20 °C, with some modifications of the procedures described previously (19.Teplitsky A. Shulami S. Moryles S. Zaide G. Shoham Y. Shoham G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 869-872Crossref PubMed Scopus (19) Google Scholar). The best crystals were obtained by mixing 5 μl of the original enzyme solution (10 mg/ml) with an equal volume of reservoir solution (containing 14% (w/v) polyethylene glycol 4000, 12% (v/v) isopropyl alcohol, and 0.1 m sodium citrate, pH 5.0–5.5) and equilibrating the drop with 1 ml of the reservoir solution. For x-ray data measurement, crystals were transferred into a cryo-solution (containing 75% reservoir solution and 25% glycerol) for about 30 s before direct flash cooling in a nitrogen gas cold stream (at 90–100 K). The diffraction pattern of all crystals grown and flash-cooled in this procedure indicated that they are isomorphous (unit cell parameters are within ±2% of the mean values) to the previously reported crystals of wild type (WT) AguA (T1 crystal form) (19.Teplitsky A. Shulami S. Moryles S. Zaide G. Shoham Y. Shoham G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 869-872Crossref PubMed Scopus (19) Google Scholar), with a tetragonal space group of P41212, mean unit cell dimensions of a = b = 74.6 Å, c = 330.3 Å, and with one molecule per crystallographic asymmetric unit. Six types of crystals were used for complete data measurement and structural analysis, as follows: 1) crystals of the selenomethionine derivative of AguA; 2) crystals of WT (uncomplexed) AguA; 3) crystals of the E285N mutant of AguA, which were co-crystallized with the substrate aldotetraouronic acid (2-O-(4-O-methyl-α-d-glucuronosyl)-β-d-xylotriose) (in the final model of this structure, only the MeGlcA group was resolved, and the xylotriose group had no apparent electron density); 4) crystals of the E285N mutant, which were soaked for 10 s in a solution containing ∼5 mg/ml of aldotetraouronic acid and then flash-cooled (in the final model of this structure, an intact substrate was found to be bound to the enzyme); 5) crystals of WT AguA, which were soaked for 5 s in a solution containing ∼4 mg/ml of aldotetraouronic acid and then flash-cooled (in the final model of this structure, the two reaction products MeGlcA and xylotriose were observed in the active site); 6) crystals of the E386Q mutant of AguA. Two independent sets of multiple anomalous diffraction data were collected on the selenomethionine crystals, using one single crystal for each multiple anomalous diffraction experiment (20.Hendrickson W.A. Ogata C.M. Methods Enzymol. 1997; 276: 494-523Crossref PubMed Scopus (326) Google Scholar). Each of these data sets included complete diffraction data that were collected at three different wavelengths around the selenium K edge (Table I). The two data sets were measured at 100 K, using a MAR-CCD (133-mm) detector (MAR-Research Inc.), on beamline BM14 of the European Synchrotron Radiation Facility (Grenoble, France). All other data sets were collected (95 K) at the National Synchrotron Light Source (NSLS) (Brookhaven National Laboratory). The WT AguA crystals were used for a 1.70-Å data set on NSLS/X8C, using a Quantum-4 CCD detector (ADSC Inc.). The E285N crystals were used for 1.85- and 1.75-Å data sets for the co-crystallized and soaked crystals, respectively, on NSLS/X12B using a Quantum-4 CCD detector. The substrate-soaked WT crystals were used for a 1.50-Å data set on NSLS/X25, using a Brandeis B4 CCD detector (Brandeis University). The E386Q crystals were used for a 2.0-Å data set on DESY/EMBL (Hamburg, Germany) beamline X13, using a MAR-CCD (165-mm) detector. All of the diffraction data sets were processed and reduced with the programs DENZO and SCALEPACK (21.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 407-426Google Scholar). Selected data collection parameters representing the nonmultiple anomalous diffraction data sets are shown in Table II.Table ISelected parameters of the MAD data measurementMAD (selenium peak)MAD (inflection)MAD (remote)Data set 1Data set 2Data set 1Data set 2Data set 1Data set 2Wavelength (Å)0.97910.979380.97890.979190.90710.88500Space groupP41212P41212P41212P41212P41212P41212Resolution (Å)2.72.82.72.82.72.8Total no. of reflections106,79190,201105,90789,642104,98089,994Unique reflections49,33644,42249,45744,14349,50248,700Completeness (last shell) (%)99.6 (100)98.4 (97.5)99.8 (100)97.7 (97.3)99.8 (100)98.7 (97.7)R-merge (last shell) (%)6.2 (24.1)10.2 (31.0)6.9 (34.2)9.6 (31.4)5.6 (23.8)9.1 (27.3) Open table in a new tab Table IIRepresentative parameters of data collection, structure determination, and refinementData setWT AguAE285N-MeGlcAE285N-substrateWT-productsE386QWavelength (Å)1.00930.97661.0391.1000.913Space groupP41212P41212P41212P41212P41212a, b (Å)73.57173.57673.55873.94673.537c (Å)329.962330.568330.010331.341328.894Resolution (Å)1.701.851.751.502.00Total no. of reflections271,828354,446664,5043,174,133887,335Unique reflections94,80872,67085,011145,97662,425Completeness (last shell) (%)93.5 (60.7)92.1 (63.1)91.6 (58.4)98.8 (90.3)99.8 (100.0) / 10.212.612.26.810.2R-merge (last shell) (%)5.7 (38.2)6.9 (23.4)6.9 (35.4)11.0 (39.9)8.3 (31.7)PDB code1K9D1K9E1K9F1L8N1MQRR-factor (R-free) (%)17.6 (20.3)19.3 (21.8)18.9 (21.1)13.9 (17.2)17.9 (20.3)No. of atomsProtein54595456545756195504Solvent650483445669466Glycerol molecules131112145Substrate/ProductNAaNA, not applicableMeGlcAMeGlcAXyl3MeGlcA, xylotrioseNARoot mean square bonds (Å)0.0060.0060.0060.0120.010Angles (units)1.3511.3511.3492.1021.468Ramachandran plotMost favored (%)91.191.090.692.190.3Allowed (%)8.48.58.57.49.2Generous (%)0.30.30.50.30.3Disallowed (%)0.20.20.30.20.2a NA, not applicable Open table in a new tab Structure Determination and Refinement—The structure of the selenomethionine derivative of AguA was solved from the anomalous signal of the 14 selenomethionine residues of the modified enzyme, using the two data sets collected on selenomethionine crystals (20.Hendrickson W.A. Ogata C.M. Methods Enzymol. 1997; 276: 494-523Crossref PubMed Scopus (326) Google Scholar, 22.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1872-1877Crossref PubMed Scopus (64) Google Scholar). Model building was performed with the program O (23.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). Due to the relatively high electron density of the selenium atoms and other landmark residues, the exact chain tracing and individual fitting of amino acid side chains was relatively straightforward. The structure of the WT AguA was solved by molecular replacement using the program AMORE (24.Navaza J. Acta Cryst. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar), with the selenomethionine AguA structure as the reference model. Further refinement was performed with the program CNS (25.Brunger 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: 905-921Crossref PubMed Scopus (16965) Google Scholar). The progress of refinement was monitored by following the overall values of Rfactor and Rfree (26.Brunger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3863) Google Scholar), calculated for 10% randomly selected reflections. All other structures were solved and refined in a similar procedure (Table II). The Overall Structure of AguA—The recombinant α-glucuronidase proteins were obtained from two related strains of G. stearothermophilus, T-1 and T-6 (AguA-T1 and AguA-T6). These two enzymes are essentially identical, differing in only 2 of the 679 amino acids of the proteins. The recombinant enzymes were overexpressed in E. coli, purified in 1-g quantities, and crystallized (16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar, 19.Teplitsky A. Shulami S. Moryles S. Zaide G. Shoham Y. Shoham G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 869-872Crossref PubMed Scopus (19) Google Scholar). Crystal structures of WT, selenomethionine derivative, catalytic mutants, and enzyme-substrate/product complexes of AguA-T1 and AguA-T6 were obtained. All of the present structures were determined at relatively high resolutions (1.5–2.0 Å) and at good final values of Rfactor (13.9–19.3%) and Rfree (17.2–21.8%) (Table I). Based on these values, the average experimental error in the coordinates of these models is around ±0.1 Å according to the Luzzati error estimation (27.Luzzati P.V. Acta Crystallogr. Sect. D. 1952; 5: 802-805Crossref Google Scholar), permitting a reliable analysis of interactions and geometries in the structures presented here. Representative sections of the electron density maps of two of the complexed structures obtained are shown in Fig. 2a. Since the structures of the WT AguA-T1 and AguA-T6 were identical within the experimental error margins (except for slight changes around residues 38 and 259, which are different between the two enzymes), we describe here only the 1.7-Å resolution structure of AguA-T1 (Fig. 2b). AguA is an α/β globular protein with overall dimensions of about 91 × 56 × 53 Å for a monomer. The enzyme is built of three distinct domains, which are connected by extended loops. The central domain has a (β/α)8 fold (TIM barrel), and it contains about half of the protein residues (amino acids 143–471). This domain is not organized as a classical TIM barrel; there are an additional two short β-strands between the first β-strand and the first α-helix (β7 and α4); the α-helix between strands β14 and β15 is missing; and two α-helices rather than one are located after both strands β11 and β15 (Fig. 2c). The N-terminal domain (residues 1–142) is made of a six-stranded β-sheet and three α-helices. The C-terminal domain, which includes the last 208 amino acids, is mostly α-helical and lies as an envelope covering almost half of the central domain (Fig. 2b). The final structural models consisted of almost all of the 679 amino acid residues of the protein, lacking only the first 2–4 N-terminal residues. In three of the five structural models, the loop containing residues 283–287 had disordered electron density, suggesting conformational flexibility. Interestingly, this loop is experimentally observed in two different conformations in the two other structures. The functional implications of this conformational change are discussed below. AguA is a homodimer in solution (16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar), yet under the conditions employed it crystallized with one molecule per crystallographic asymmetric unit. Generation of all of the symmetryrelated molecules in the crystallographic unit cell revealed a possible dimeric form, which seems to be the most biologically relevant. The contact region between the two subunits involves the following residues from each monomer: Trp328 and Arg329 from the (β/α)8 domain and Glu536, Arg548, Glu654, Asp657, Arg665, and Lys666, from the C-terminal domain (Fig. 3). The assignment of this dimerization contact is supported by mutagenesis and biochemical studies, showing that these residues are essential for dimer formation. 2D. Shallom, G. Golan, G. Shoham, and Y. Shoham, manuscript in preparation. Comparison with Family 67 and Other Glycosidases—To date, the structure of GlcA67A from C. japonicus is the only other three-dimensional structure of a GH-67 glycosidase (9.Nurizzo D. Nagy T. Gilbert H.J. Davies G.J. Structure. 2002; 10: 547-556Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28.Nagy T. Nurizzo D. Davies G.J. Biely P. Lakey J.H. Bolam D.N. Gilbert H.J. J. Biol. Chem. 2003; 278: 20286-20292Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). AguA and GlcA67A share sequence identity of 42% and similarity of 59% of 665 aligned residues. The overall fold of the two enzymes is similar, and structure comparison shows high resemblance with root mean square deviations of 1.13 Å for all C-α atoms (structural homology was performed using the DALI server (29.Holm L. Sander C. Nucleic Acids Res. 1998; 26: 316-319Crossref PubMed Scopus (596) Google Scholar)). The two structures differ mainly in the C-terminal domain, where GlcA67A has an additional polypeptide chain of 33 residues, which is not present in the AguA sequence. The most striking difference between the two enzymes is in their dimeric structure. Whereas in AguA the contact region between the two monomers is relatively narrow and located at the tip of the C-terminal domain, in GlcA67A the dimer-forming residues are scattered on a much wider surface, which encloses parts from the C terminus as well as the middle of the (β/α)8-barrel (9.Nurizzo D. Nagy T. Gilbert H.J. Davies G.J. Structure. 2002; 10: 547-556Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The dimeric interfaces of the two enzymes do not overlap. As a result, in GlcA67A the dimeric interface is closer to the active site than in AguA, but in both enzymes the substrate binding sites are exposed to the solvent. The difference in the dimeric forms of AguA and GlcA67A is consistent with the degree of sequence conservation of the two α-glucuronidases (see Supplementary Fig. 1). Although the (β/α)8-barrel is one of the most common folds for enzymes in general and glycosidases in particular, the overall arrangement of the three domains comprising the α-glucuronidases seems to be unique to GH-67 glycosidases. The combination of the N-terminal domain and the central domain of the AguA structure resembles domains I and II of the Streptomyces plicatus β-hexosaminidase (30.Mark B.L. Vocadlo D.J. Knapp S. Triggs-Raine B.L. Withers S.G. James M.N. J. Biol. Chem. 2001; 276: 10330-10337Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and domains II and III of Serratia marcescens chitobiase (31.Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (326) Google Scholar). Superpositions reveal relatively low structural similarities, with average root mean square differences of 1.9 and 2.5 Å, between the AguA domains and the equivalent N-terminal and TIM-barrel domains, respectively. As in these GH-20 glycosidases, the function of the N-terminal domain of AguA is not yet clear. Previously, we have shown that removal of this domain does not significantly affect thermostability, but it does prevent the correct oligomerization of the protein and lowers its catalytic activity to 1% of the wild type activity (16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar). Interestingly, from the structure it seems that this domain is quite distant from the dimerization area, and its removal probably exposes hydrophobic residues located on the (β/α)8 domain, causing the altered oligomerization observed. Structures of AguA Complexed with Intact and Cleaved Substrates—Based on biochemical and mutational studies of AguA, it was previously suggested that the active site of the enzyme is located at the middle (β/α)8 domain and that the conserved residues Glu285, Asp364, and Glu392 have an important role in catalysis (16.Zaide G. Shallom D. Shulami S. Zolotnitsky G. Golan G. Baasov T. Shoham G. Shoham Y. Eur. J. Biochem. 2001; 268: 3006-3016Crossref PubMed Scopus (46) Google Scholar). From the structure of the WT AguA, it appears that Asp364 and Glu392 are located at the C-terminal side of the β-sheets of the (β/α)8 domain (Fig. 4a). In an attempt to trap the Michaelis complex, we used the catalytic mutant E285N together with the natural substrate aldotetraouronic acid (2-O-(4-O-methyl-α-d-glucuronosyl)-β-d-xylotriose, or MeGlcAX-yl3 for short). Co-crystallization experiments have resulted in the 1.85-Å resolution binary complex of the enzyme with the reaction product MeGlcA, indicating that the residual activity of this mutant during the crystallization and the time elapsed until the data collection (about 2 months) was enough to cleave the substrate (Fig. 4b). When the E285N crystals were briefly soaked in the MeGlcAXyl3 solution followed by flash freezing, the electron density at 1.75-Å resolution unambiguously showed the intact substrate bound to the enzyme (Figs. 2a and 4c). When a similar strategy of brief soaking and flash freezing was employed with the WT enzyme, the 1.5-Å resolution structure revealed that the substrate was cleaved, and the two reaction-products, MeGlcA and xylotriose, are trapped in the active site (Figs. 2a and 4d). In the two structures where the xylotriose moiety is present (E285N-substrate and WT-products), only the first two sugar units of the xylotriose could be resolved in the models, and the reducing end xylose unit is probably exposed to the solvent and too loose to produce clear electron density. All of the observed ligands are found bound at the active site of the enzyme, which has a pocket topology of exo-acting glycosidases (Fig. 4e) (32.Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar). In the structure of the WT enzyme, a glycerol molecule (originating from the cryogenic freezing solution) is located in the active site, probably replacing water molecules present in these positions in aqueous solutions (Fig. 4a). Electrostatic potential analysis of the solvent-accessible surface of the protein indicates that the active site cavity is relatively polar, with a distinct positively charged region at the glucuronic acid binding site. In all of the complexed structures, the MeGlcA molecule is bound in a similar manner at the bottom of the active site pocket and held in place by stacking interactions with the conserved Trp150 and an extensive hydrogen-bonding network with the conserved residues Glu158, Arg159, Asn201, Lys281, Arg318, Arg335, Lys359, Asp364, and Glu392 (Fig. 4, b–d). Trp150 is also involved, together with the conserved Val200, in forming the hydrophobic surrounding around the methyl group of the MeGlcA, as was observed in the GlcA67A from C. japonicus (28.Nagy T. Nurizzo D. Davies G.J. Biely P. Lakey J.H. Bolam D.N. Gilbert H.J. J. Biol. Chem. 2003; 278: 20286-20292Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In both the substrate and the product complexes, the conserved Trp540 forms stacking interactions with the xylose at the +1 subsite. Compared with the MeGlcA moiety, all of the xylose units are more exposed to the solvent and share fewer interactions with the enzyme. In glycosidases in general, the sugar at the -1 subsite (the glycon) is bound by a larger number of interactions as compared with the +1 subsite (the aglycon). This is consistent with the observation that the specificity of glycosidases is governed mainly by th
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