Crystal Structures of Geobacillus stearothermophilus α-Glucuronidase Complexed with Its Substrate and Products

Abstract

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. 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. G. Shoham G. Henrissat B. T. Shoham Y. 2003; PubMed Scopus Google Scholar, T. S. T. Shoham G. Shoham Y. 2001; PubMed Scopus Google Scholar, C. Shoham Y. Appl. Environ. Microbiol. PubMed Google Scholar, D. D. Shoham G. T. Shoham Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The α-glucuronidase T-6 (AguA) is part of a in the of acid S. Shoham Y. J. 1999; PubMed Google Scholar, G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). Here we high resolution crystal structures of AguA in complex with substrates and products. structures for several that the catalytic mechanism of AguA a of the loop carrying the general acid a distortion of the substrate into a conformation closer to the conformation required for the oxocarbenium ion-like transition and charge between the substrate and the general acid so as to allow its protonated The structural with of catalytic that the two carboxylic residues Asp364 and Glu392, as general together the nucleophilic water molecule. and α-glucuronidases from G. stearothermophilus T-6 and T-6 and AguA and and in The of the wild and enzymes was as described G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). of the of AguA was carried out in the strain as described T. Shoham G. Shoham Y. J. Biotechnol. 2000; PubMed Scopus Google and as for the wild was by the of from acid the and for Y. G. Res. Scopus Google as described G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). and the and at with some of the described S. S. G. Shoham Y. Shoham G. Biol. 1999; PubMed Scopus Google Scholar). The by 5 of the enzyme with an of and and the with of the data into a and for in a The of all and in this that are cell are of the to the of wild AguA crystal S. S. G. Shoham Y. Shoham G. Biol. 1999; PubMed Scopus Google with a of cell of a and with one molecule of used for data and structural as of the of of of the of AguA, which with the substrate acid the of this the was and the of the which for in a of acid and the of this an intact substrate was found to be to the of AguA, which for 5 in a of acid and the of this the two products and in the active of the of of data on the one crystal for 1997; PubMed Scopus Google Scholar). of these data data that at three different the The two data at a on of the Synchrotron other data at the National Synchrotron Light National The AguA used for a data on a The used for and data for the and on a The used for a data on a The used for a data on a of the data and with the and 1997; Scholar). data representing the data are in of the data of in a new of data structure and of of bonds in a new and structure of the of AguA was from the of the residues of the the two data on 1997; PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus Google Scholar). was with the PubMed Scopus Google Scholar). to the high of the and other residues, the and of acid was The structure of the AguA was by the J. Scopus Google with the AguA structure as the was with the P. J. M. T. Biol. 1998; PubMed Scopus Google Scholar). The of was by the overall of and PubMed Scopus Google for other structures and in a The of α-glucuronidase from two of G. stearothermophilus, and T-6 and two enzymes are in of the of the The enzymes in in and G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar, S. S. G. Shoham Y. Shoham G. Biol. 1999; PubMed Scopus Google Scholar). structures of WT, catalytic and complexes of and of the structures at high and at of and on these the in the of these is to the D. Google a of and in the structures of the of two of the structures are in the structures of the and the for residues and which are different between the two we describe the resolution structure of (Fig. AguA is an with overall of for a The enzyme is of three domains, which are by The central has a fold and it of the residues is as a are an two short between the first and the first and the between and is and two than one are and (Fig. The is of a and three The which the acids, is and as an of the central (Fig. The structural of all of the acid residues of the the first In three of the structural the loop residues this loop is in two different conformations in the two other structures. The of this are AguA is a in G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google the it with one molecule of all of the in the cell a which to be the most The between the two the residues from and from the and and from the (Fig. The of this is by and that these residues are for G. G. and Y. in with 67 and date, the structure of from C. japonicus is the other structure of a (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, T. D. Davies G.J. P. Gilbert H.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). AguA and of and of of The overall fold of the two enzymes is and structure high with of for all was the server C. Res. 1998; PubMed Scopus Google The two structures in the has an of residues, which is in the AguA The most between the two enzymes is in structure. in AguA the between the two is and at the of the in the residues are on a which parts from the as well as the 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 of the two enzymes a in the is closer to the active site than in AguA, but in enzymes the substrate are to the The in the of AguA and is with the of of the two α-glucuronidases 1). the (β/α)8-barrel is one of the most for enzymes in general and glycosidases in the overall of the three domains comprising the α-glucuronidases to be to The of the and the central of the AguA structure domains and of the S. Withers S.G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and domains and of Struct. Biol. PubMed Scopus Google Scholar). structural with of and between the AguA domains and the and domains, in these glycosidases, the function of the of AguA is we that of this but it the of the and its catalytic to of the wild G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). from the structure it that this is from the and its residues on the the of AguA with and on and of AguA, it was that the active site of the enzyme is at the and that the residues and an in catalysis G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). From the structure of the AguA, it that Asp364 and are at the of the of the (Fig. In an to the we used the catalytic together with the natural substrate acid or for in the resolution complex of the enzyme with the MeGlcA, that the of this the and the the data was to cleave the substrate (Fig. the in the by the at resolution the intact substrate to the enzyme and a of and was with the the resolution structure that the substrate was and the two and are in the active site and In the two structures the is and the first two sugar of the could be resolved in the and the xylose is to the and to of the are found at the active site of the which has a of glycosidases (Fig. G. Henrissat B. Structure. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In the structure of the a molecule from the is in the active site, water in these in (Fig. of the of the that the active site is with a at the acid site. In all of the the molecule is in a at the of the active site and in by with the and an extensive with the residues and (Fig. is together with the in the the of the MeGlcA, as was in the from C. japonicus T. D. Davies G.J. P. Gilbert H.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the substrate and the the with the xylose at the with the all of the xylose are more to the and with the In glycosidases in the sugar at the is by a larger of as with the is with the that the of glycosidases is by the of the sugar in the the catalytic involving the anomeric carbon In the the three sugar and two are in the conformation, that the products are in the active site in an in the complex of with the intact substrate, the in is distorted from the conformation and a conformation (Fig. In this conformation, the is in the conformation of the MeGlcA, this is in the the sugar ring at the is closer to the conformation required for the oxocarbenium ion-like transition In the structure of the a water molecule is found between Glu392, and the anomeric carbon of the substrate, from the carboxylic of Asp364 and and from the anomeric carbon and that the water molecule act as the nucleophilic the anomeric carbon by the general is which of the residues, Asp364 or Glu392, the of the the loop residues is in the free enzyme and the two complex but it is well in the structure of the enzyme in complex with the products. In its the loop is in a “closed” conformation, and Glu285 is in an position to function as the general acid from the of the xylose at the is the glycosidic bond of this structure with the complex structure that Glu285 is from the glycosidic of the substrate, a most for the of the (Fig. it that the of the in the active site this loop and Glu285 in a position that is a in and its to to a of in the catalytic of AguA G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). from the it is that this has with either of the catalytic residues, and its to the substrate, between its carboxylic and the carboxylic of the MeGlcA, is in the structure the loop is well but it a different conformation with the structure of the The loop is from the (β/α)8-barrel into a position that Glu285 from its in the wild structure (Fig. is to that this conformation is one of the conformations that this loop in the free to this loop in its “open” in the structure is a of from the of two bonds between was to and to the of to the the loop be in its of the catalytic residues of glycosidases is by the of enzymes in which the are by The are to substrates different with such as and of the and the wild enzymes Withers S.G. 1999; PubMed Scopus Google Scholar). In glycosidases, the of the nucleophilic can be by the J. Withers S.G. 2002; PubMed Scopus Google Scholar). AguA and most other α-glucuronidases are in substrates such as or G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar, S. 1995; PubMed Scopus Google Scholar, T. Y. Biotechnol. 56: Scopus Google Scholar, M. M. J. Biotechnol. 2000; PubMed Scopus Google Scholar, Appl. Microbiol. Biotechnol. 1995; PubMed Scopus Google the of the catalytic residues of this family a for the catalytic residues is by structural of complexes with and transition state Inverting glycosidases the glycosidic bond two carboxylic acids, and the active site is from in The general catalyst is a nucleophilic water molecule that the anomeric carbon of the sugar from the of the glycosidic The other carboxylic as a general acid the G. Withers S.G. Scholar, Chem. Scopus Google Scholar). The crystal structures of AguA in complex with its substrate and products that the three and Glu392, are to the glycosidic bond and can act as the catalytic In the Asp364 and are on the of the sugar in an position to activate a nucleophilic water molecule and act as the general catalytic a water between Asp364 and was in the structure of the complex (Fig. of two residues with a water molecule between was in the structure of the α-glucuronidase from C. japonicus (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, T. D. Davies G.J. P. Gilbert H.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the water molecule is in an from the two residues, it to on structural data which of is the catalytic In the AguA the between the two carboxylic of Asp364 and and the of the water molecule is a for a of the water molecule by these The of Asp364 and are a from and and of bonds and (Fig. are to the of Asp364 and Glu392, in with as the catalytic we that the of either Asp364 or into residues in a of 5 of in the catalytic G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). together the structural and we that Asp364 and are the nucleophilic water and of could be as the catalytic residues of site in which two residues act together as general was in other inverting glycosidases, the from the S. S. R. R. J. Biol. 1997; PubMed Scopus Google Scholar) and the from and Y. S. Visser J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. Y. Visser J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. T. 2002; PubMed Scopus Google Scholar). The of Glu285 as the catalytic general acid of AguA to be more it is a bond from the glycosidic and can as the the of the loop carrying as in the different AguA is the catalytic residues are part of an extensive that the position and the charge for catalysis G. Withers S.G. 2001; PubMed Scopus Google Scholar). are and structural that substrate and the position of the catalytic residues in glycosidases C. J. Biol. 2003; PubMed Scopus Google Scholar, M. Davies G.J. 1999; PubMed Scopus Google Scholar, R. M. J. Biol. PubMed Scopus Google Scholar). in the of the from C. the loop is to Glu285 in is in in the free and of the enzyme (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, T. D. Davies G.J. P. Gilbert H.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In it is in an to the “closed” conformation of the loop of The state of Glu285 is of In to as a general acid and a to the this to be protonated in the structure of AguA in complex with its products the loop is in its “closed” it that the of Glu285 to its than The of is from the of and a short bond is to the of Glu285 by to the of Glu285 by the state (Fig. can the required of Glu285 is the acid of the substrate The of the is from and could the charge of the In the between Glu285 and the of the is between these two negative In several other inverting glycosidases, other residues are found in the proximity of the catalytic and can to its Y. S. Visser J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, G. T. M. M. D. G.J. M. J. J. Am. Chem. Soc. 2002; PubMed Scopus Google Scholar, R. Structure. Full Text Full Text PDF PubMed Scopus Google Scholar). in glycosidases, the catalytic residues are 5 and the charge of the nucleophilic and was to the cycle of the acid/base G. M. Withers S.G. PubMed Scopus Google Scholar). is likely that in the of the substrate the of the general acid in and the protonated which is required for its is a mechanism, in which the of the substrate catalysis by the charge of a catalytic representing a new variation of substrate-assisted catalysis mechanism P. 2000; PubMed Scopus Google Scholar). described in the complex of with the intact substrate, the is found in a conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. in the structure of GlcA67A, the of the substrate is found in the conformation T. D. Davies G.J. P. Gilbert H.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of substrates to conformations that the transition state is a in structures G.J. D.L. Soc. 2003; PubMed Google Scholar, Davies G.J. M. Curr. Opin. Chem. Biol. 2002; 6: PubMed Scopus (360) Google Scholar). are sugar ring which the and of a in one as required for the oxocarbenium ion-like transition the and and the and half-chair to the of described by Davies G.J. D.L. Soc. 2003; PubMed Google is from the distortion of the in AguA into one of these transition state the structure is the complex but a stable between the state and the that the complex is via the this from the conformation of the is a for the for the transition The was for two other inverting glycosidases, the J. G. Gilbert H.J. Davies G.J. Chem. 2003; Scopus Google Scholar) and the M. P. J. Biol. 2002; PubMed Scopus Google as well as for the xylanases G. M. M. C. Davies G.J. Chem. Biol. 1999; 6: Full Text PDF PubMed Scopus Google Scholar, G. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is the general mechanism of inverting glycosidases an water the anomeric the for a nucleophilic in In AguA other of family 67 glycoside is more for are than The by the of Asp364 and Glu392, the nucleophilic and the anomeric is to an between of the two and the acid catalyst In the catalytic mechanism of AguA is to as (Fig. the substrate is into the active site and the “closed” conformation of the The sugar ring in the is distorted into a conformation, and the negative charge of the acid the of the Asp364 and activate the nucleophilic water which the anomeric carbon of the substrate, and Glu285 its to the glycosidic The hydrolysis an oxocarbenium transition in which the sugar ring in the is likely to be distorted into a the products are from the active site in an in which the the G. D. S. G. G. T. Shoham G. Shoham Y. J. 2001; PubMed Scopus Google Scholar). The of the enzymatic is well in the structures of the AguA complexes described The bond between the and the in the intact substrate is in an in the complex of the enzyme with its the into a configuration (Fig. The sugar ring is stable in the configuration and into the more stable as in the complex of with the enzyme has a higher to the in the which into the active site at In of these the well with of glycoside hydrolases as inverting enzymes P. de Vries R.P. Vrsanska M. Visser J. Biochim. Biophys. Acta. 2000; 1474: 360-364Crossref PubMed Scopus (43) Google Scholar). and Synchrotron for in the data with