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J. Biol. Chem., Vol. 281, Issue 34, 24922-24933, August 25, 2006
Crystal Structures of Clostridium thermocellum Xyloglucanase, XGH74A, Reveal the Structural Basis for Xyloglucan Recognition and Degradation* 1 1![]() ![]() ![]() ![]() 3
From the
Received for publication, April 13, 2006 , and in revised form, June 12, 2006.
The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.
Plant cell wall polysaccharides are the most abundant carbohydrate polymers in nature and constitute an important renewable natural source of energy available for conversion to biofuels (1). The plant resource is, however, difficult to exploit primarily because its components are extremely resistant to degradation; plant cell wall polysaccharides are often present as insoluble, often cross-linked, structures in which cellulose is the most abundant component. In the cell wall of flowering plants, cellulose is cross-linked by two major types of glycan: xyloglucans and glucuronoarabinoxylans. The xyloglucans form a complex network of hydrogen-bonded interactions with cellulose microfibrils that confers rigidity and extensibility to the walls of all dicotyledons and about one-half of monocotyledons (2). The xyloglucan polysaccharide consists of a linear chain of -1,4 D-glucan regularly substituted with -1,6 D-xylosyl units, which is, in a species-dependent manner, further derivatized with -L-arabinose or -D-galactose (2, 3), Fig. 1. In primary cell wall xyloglucans, the first galactose moiety in the oligosaccharide repeat is commonly substituted with -1,2 L-fucose (4). Considerable interest in the structure, biosynthesis, and enzymatic modification of xyloglucans has been sustained because of the important role these polysaccharides play in plant cell wall morphogenesis (4-8), as well as the emerging technical applications of xyloglucans in food products, pharmaceutical delivery (9, 10), cellulose fiber modification (11-13), and biofuel production (1).
Microorganisms have evolved sophisticated mechanisms to degrade plant cell wall polysaccharides and consequently exploit this rich carbon and energy source. Aerobic bacteria and fungi secrete several individual enzymes that synergistically degrade plant cell walls (14). Some anaerobic microorganisms, notably Clostridia, utilize a large multi-enzymatic complex called the cellulosome (15). The cellulosome displays a consortium of hydrolytic plant cell wall degrading enzymes, which may change with time, including cellulases, hemicellulases, pectinases, and various esterases. The Clostridium thermocellum (Ct)4 cellulosome is one of the best studied cellulosome systems. This cellulosome is a multiprotein complex of about 3 MDa and displays endoglucanase, cellobiohydrolase (exoglucanase), xylanase, chitinase, and
There is particular interest in the exploitation of the cellulosome from C. thermocellum, primarily because of the potential it offers for the degradation of lignocellulosic waste and subsequent generation of ethanol (reviewed in Ref. 1). To date, 71 open reading frames have been identified as cellulosomal components in Ct and about 23 genes can be ascribed a direct role in cellulose hydrolysis (18). About half of the other proteins in the cellulosome are described as hemicellulolytic enzymes, highlighting the importance of these accessory enzymes in the processing of cellulosic composites. Recently, two major enzymes implicated in hemicellulose degradation by the Ct cellulosome have been characterized; an endo-
Many xyloglucanases have been classified into glycosyl hydrolase family 74 (hereafter GH74) in the Carbohydrate Active Enzyme (CAZy) classification (20); recently reviewed in Ref. 21. From the structural point of view, the only member of the GH74 family published to date is the reducing end-specific cellobiohydrolase (OXG-RCBH) from Geotrichum sp. M128 (22). OXG-RCBH recognizes the reducing end of various xyloglucan-derived oligosaccharides and releases two glucosyl residues of the type GG, XG, or LG (nomenclature according to Ref. 23, see also Fig. 1) suggesting the presence of at least four subsites (24). Additionally, it was noted that the glucosyl residue at position +2 (nomenclature according to Ref. 25) has to be unsubstituted, while that at -1 should preferentially posses a xylosyl substituent. The OXG-RCBH structure consists of a tandem repeat of two seven-bladed -propeller motifs with the catalytic center formed by the interface of these two domains. The recent enzymatic characterization of the endoxyloglucanase, Xgh74A, shows that the enzyme hydrolyzes the glycosidic bond of the unbranched glucosyl residues in xyloglucan, to yield XXXG, XLXG (or XXLG), and XLLG oligosaccharides (19). Here we describe the crystal structure of Xgh74A both as an uncomplexed apoenzyme at 2.1 Å resolution, and that of an inactive variant, Xgh74A-D70A, with a single molecule of XLLG and one of XXLG bound in the active site cleft. These structures, in light of kinetic and hydrolysis data, reveal the specificity determinants responsible for xyloglucan recognition and provide insight into the hydrolysis of this important plant cell wall polysaccharide.
Cloning of Ct Xgh74A Catalytic Domain The cellulosomal xyloglucanase A from C. thermocellum is a bi-modular enzyme containing an N-terminal family 74 glycoside hydrolase (GH) catalytic domain followed by C-terminal dockerin (19). To express the xyloglucanase catalytic module in Escherichia coli, Xgh74A hereafter, the DNA fragment encoding the protein domain was amplified by PCR from C. thermocellum YS genomic DNA with the thermostable DNA polymerase Pfu Turbo (Stratagene). The primers, 5'-CTCGCTAGCATTTCCAGCCAGGCTGTA-3' and 5'-CACCTCGAGATCTGAAGCAGGTTCGCC-3', incorporated NheI and XhoI restriction sites, which are depicted in bold. The amplified product was ligated into pMOSBlue (Amersham Biosciences) and sequenced to ensure that no mutations had occurred during the polymerase chain reaction. The recombinant pMOSBlue derivative was digested with NheI and XhoI, and the excised Xgh74A encoding gene was cloned into the similarly restricted expression vector pET21a to generate pCG1. CtXgh74A encoded by pCG1 contains a C-terminal His6-tag.
Site-directed Mutagenesis
Production of Recombinant Xgh74A and Mutants
High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) Gradient A (for Analysis of Glc4-based Xylogluco-oligosaccharides)Solvent A: 100 mM NaOH, solvent B: 100 mM NaOH 200 mM NaOAc. Gradient program: 0-4 min, 100 mM NaOH, 60 mM NaOAc; 4-10 min linear gradient of 60 mM NaOAc to 117 mM NaOAc; 10-11 min 200 mM NaOAc. The system was then re-equilibrated for 4 min with the initial conditions prior to the next injection. Gradient B (for Analysis of Higher Order Xylogluco-oligosaccharides)Solvent A: 100 mM NaOH, solvent B: 100 mM NaOH 500 mM NaOAc. Gradient program: 0-3 min, 100 mM NaOH, 40 mM NaOAc; 3-11 min linear gradient of 40 mM NaOAc to 170 mM NaOAc; 11-18 min. linear gradient 170 mM NaOAc to 200 mM NaOAc; 18-19 min 500 mM NaOAc. The system was then re-equilibrated for 4 min with the initial conditions prior to the next injection.
Mass Spectrometry of Xylogluco-oligosaccharides
Preparation of Xylogluco-oligosaccharides (XGOs) from Deoiled Tamarind Kernel Powder
XXXGXXXG and Partially Degalactosylated Glc12-based Xyloglucan OligosaccharidesDegalactosylated higher order oligosaccharides were produced exactly as described in the preceding paragraph, except that 4 units of
Crystallization, Data Collection, Structure Solution, and Refinement
Although SeMet data were collected, Xgh74A structure was solved by molecular replacement because the structure of the homologous Geotrichum sp. M128 oligoxyloglucan reducing end-specific cellobiohydrolase (PDB ID: 1SQJ) became available at the time these studies were carried out. The program PHASER (29) was used to place the 4 molecules of Xgh74A in the asymmetric unit. The Xgh74A model was built and refined, against the SeMet data using Arp-wARP (28). Subsequently, the refined SeMet apo enzyme structure was used as a molecular replacement model for the solution of the Xgh74A-D70A ligand complex, which crystallized in a P43212 form with 2 molecules in the asymmetric unit. Manual rebuilding and ligand placement were carried out with COOT (30). Solvent model was build with Arp-wARP with maximum likelihood refinement using REFMAC (31).
Activity of Xgh74A on Xyloglucan Limit Digest of Tamarind Xyloglucan and Glc4-, Glc8-, and Glc12-based Xylogluco-oligosaccharides by Xgh74ATamarind xyloglucan (1 g/liter) and Xgh74A (0.02 mg/liter) was incubated in 20 mM potassium phosphate buffer pH 7.0 (total volume 25 µl) at 50 °C for 30 min. The reaction was stopped by incubation at 95 °C for 10 min. 10 µl of the sample was analyzed by HPAEC-PAD (gradients A and B). A mixture of Glc4-based xylogluco-oligosaccharides (XXXG, XLXG, XXLG, XLLG) (3.5 g/liter) and Xgh74A (2.3 g/liter) were incubated in 20 mM potassium phosphate buffer pH 7.0 (total volume 25 µl) at 50 C for 30 min. The reaction mixture was then diluted 1:100 into ultrapure water and directly injected for HPAEC-PAD analysis (gradient A). Glc8-(6.3 g/liter) or Glc12-based (9.5 g/liter) xylogluco-oligosaccharides and Xgh74A (0.02 g/liter) were incubated in 20 mM potassium phosphate buffer pH 7.0 (total volume 25 µl) at 50 °C for 30 min. The reaction mixture was then diluted 1:100 into ultrapure water and directly injected for HPAEC-PAD analysis (gradients A and B). For each of the above cases, comparative experiments were carried out by substituting Trichoderma longibrachiatum endoglucanase (0.25 units, EGII, lot 50201, Megazyme, Erie,) for Xgh74A under identical conditions.
The Ct -1,4-xyloglucan hydrolase Xgh74A is an 842-residue protein that consists of a N-terminal catalytic module (residues 1-776) and a C-terminal dockerin module (residues 777-842), Fig. 2B. For structural and kinetic studies, the Xgh74A catalytic module was cloned into the pET21a vector from residue Ile28 to Glu762 and overexpressed in E. coli Tuner cells (Novagen). Kinetics and Specificity of Xgh74AXgh74A was active on tamarind xyloglucan, lichenan, and the artificial substrates carboxymethyl cellulose (CMC 4M, Megazyme, Eire) and hydroxyethylcellulose, Table 1. Xgh74A shows considerably greater catalytic efficiency on xyloglucan (kcat/Km 63 (g/liter)-1 min-1) than on any of the other substrates including CMC 4M (kcat/Km 9 (g/liter)-1 min-1), which like xyloglucan is substituted at the 6 position, in this case with an average degree of substitution of 4 carboxymethyl groups every 10 sugars. The enzyme showed no activity on xylan, polygalacturonic acid, wheat arabinoxylan, rhamnogalacturan, curdlan, laminarin, galactomannan, galactan, and arabinan with a small unquantifiable activity on glucomannan. Taken together the results are strongly suggestive that Xgh74A is a true xyloglucanase.
Limit digest analysis of tamarind xyloglucan hydrolysis by Xgh74A indicates the liberation of XXXG, XLXG, XXLG, and XLLG (Fig. 3) as the major products. Longer oligosaccharides did not accumulate, but were observed as transient species by HPAEC-PAD. In contrast to previous work on this enzyme (19), we saw no evidence for the production of XXG, XXX, or XXGG by mass spectrometry (Fig. 4). We speculate that the acidic ESI conditions and/or different ion optics employed by Zverlov et al. (19) may have contributed to the formation of fragment ions in the ESI source. Here, our use of MeOH/H2O/NaCl as an ESI solvent minimized oligosaccharide fragmentation and yielded exclusively [M+Na]+ or [M+2Na]2+ adducts, depending upon the applied cone voltage (Fig. 4).
To investigate whether Xgh74A cleaves tamarind xyloglucan at a position other than the anomeric carbon of the unbranched glucosyl moiety (Fig. 1), limit digest experiments were performed on xylogluco-oligosaccharides based on Glc4-, Glc8-, and Glc12 backbones. Cleavage of the Structure of C. thermocellum Xyloglucanase Xgh74AThe structure of the catalytic module of Xgh74A was solved in a P21 crystal form by molecular replacement using the homologous Geotrichum sp. M128 oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH, PDB ID: 1SQJ) as a search model. The final Xgh74A model, an apoenzyme incorporating SeMet in place of methionine, comprises residues Val33 to Ser760 and was refined to crystallographic R-factors of 17.5% (Rcryst) and 21.0% (Rfree) with diffraction data to a resolution of 2.1 Å. Data collection and refinement statistics are summarized in Table 2. The asymmetric unit contains 4 copies of the polypeptide chain that can be superimposed with an average root-mean-square deviation (RMSD) of 0.25 Å showing no significant conformational differences due to crystal packing. Some of the contacts between molecules in the asymmetric unit are mediated through the coordination of cadmium ions (added as a crystallization component).
Xgh74A consists of two sevenbladed -propeller domains (Fig. 5A), as expected from the sequence homology with OXG-RCBH. The N-terminal domain of Xgh74A comprises residues 63 to 459 while the C-terminal domain involves residues 33-62 and 460-760. Similarly to OXG-RCBH, the Xgh74A N-terminal domain is orientated at angle of 90 degrees relative to the C-terminal domain and interactions between these domains occur primarily through H-bonding and hydrophobic interactions over a shared contact area of about 7530 Å2. The N- and C-terminal domains, which can be superimposed with an RMSD of 3.1Å, exhibit 19% sequence identity, which most likely reflects an ancient gene duplication event. The Xgh74A N- and C-terminal domains are connected by two loop segments, one located in the N terminus and the other in the middle of the sequence. In the OXG-RCBH structure, the N- and C-terminal domains are linked by three segments; one in the N terminus, the second in the middle and the third in the C terminus of the sequence (this C-terminal segment adds a fifth strand to the second blade of the N-terminal propeller, which is absent in Xgh74A).
The overall topology of Xgh74A is thus very similar to OXG-RCBH with all the secondary structure elements linked through identical connectivity. The C traces superimpose with a RMSD of 1.8 Å for 664 equivalent residues, reflecting 39% sequence identity (calculation performed with DALI, Ref. 34). Not surprisingly, greater structural divergence is found in the loops connecting the blades of the -propeller architecture. The most important of these differences is the different conformation adopted by the loop Thr397-Pro406 in Xgh74A compared with its structural equivalent Asn374-Thr391 in OXG-RCBH, which may contribute to the significantly different substrate specificity of the two enzymes. These structural details are discussed, below, in the light of the ligand complexes of Xgh74A.
Active Site StructureIt is immediately apparent (Fig. 5) that the substrate binding region of Xgh74A lies in an open cleft. This groove is formed at the intersection of the N- and C-terminal domains. The surface of this cleft is formed by the loops connecting the
Catalysis by family GH74 enzymes occurs with inversion of anomeric configuration; i.e. the stereochemistry of the product is inverted with respect to the
Structure of the Xgh74A-D70A Mutant in Complex with Glc4-based Xylogluco-oligosaccharidesTo probe the structural determinants of xyloglucan recognition we attempted to cocrystallize Xgh74A and two inactive Xgh74A variants (D70A and D480A) with preparations of xylogluco-oligosaccharides based on Glc4 backbones. Crystals of the Xgh74A-D70A mutant, complexed with a mixture of Glc4-based oligosaccharides, were obtained by co-crystallization and diffracted to 1.95-Å resolution. Crystals were indexed in the P43212 space group and contained two molecules in the asymmetric unit. The complex structure is essentially identical to the ordered parts of the apo structure resulting in an RMSD value of 0.3 Å for the C atoms. In the ligand complex structure, however, it is also possible to build the previously disordered loops, which all interact directly with the bound oligosaccharide.
The electron density map displays well defined density for seventeen sugar rings, corresponding to a molecule of XLLG and another of XXLG, either side of the catalytic center (Fig. 6). The two molecules sit in an extended conformation at the bottom of the cleft on both sides of the catalytic residue Asp480.It is possible that the desolation afforded by ligand binding contributes to the pKa elevation of the catalytic acid. The glucosyl backbones extend about 20 Å in opposite directions from the center point described by the Asp480 residue. The interaction of the two ligand molecules extends over an area of
Despite co-crystallization with a mixture of oligosaccharides (in relative proportions XXXG 2: XLXG 1: XXLG 3: XLLG 3,) the species that we observe bound to the enzyme corresponds to XLLG in the "minus" subsites (subsite nomenclature according to Ref 25.) and XXLG in the "positive" leaving group subsites. Thus, four
At subsite -2, the plane of the Glc-2 ring is rotated 180 degrees relative to the plane of Glc-1 contacting the enzyme through two H-bonding interactions mediated by Glc2- O2 and O3 with the side chain of Asn749. Xyl-2' ring is located almost parallel to the plane of Tyr214 aromatic ring and its interaction with the protein is mediated by two H-bonds between Xyl-2' O4 and the side chain oxygen of Tyr295 and side chain NH1 of Arg158. Gal-2'' does not contact the protein directly but forms an H-bond interaction with a water molecule which in turn interacts with the main chain nitrogen atom of Tyr214. The Gal-2'' moiety does not appear to be specifically recognized by the enzyme; instead it is located close to the border of the cleft and exposed to the solvent.
The -3 subsite glucoside is "stacked" on the side chain of Trp125 and does not form any hydrogen-bonding interactions with the enzyme. The Xyl-3' ring in Xgh74A describes an angle of
In the positive or "leaving group" subsites of Xgh74A, a second XXLG molecule binds in an extended conformation in which an imaginary line drawn through the glucosyl residues at the positive subsites makes an angle of ca. 60 degrees with respect to the trajectory of the glucosyl backbone bound in the negative subsites; i.e. the chain is bent. The interaction area with the enzyme is smaller (154 Å2) than the area of contact over the minus subsites. Direct H-bonding contacts between this molecule and the protein atoms are also fewer and are confined just to the +1 site. The catalytic acid, Asp480, contacts the glucosyl unit at the +1 and -1 sites forming H-bonding interactions with the O4 of Glc+1 unit consistent with its role in aiding departure of this group, through proton donation, during catalysis. The +1 sugar residue also interacts with the aromatic nitrogen of Trp410 (O2) and the main chain oxygen of Gly430. Asp524 forms two H-bonding interactions with O2 and O3 of the Xyl+1' residue and additionally, the O5 atom of this residue contacts the O3 of Xyl+2'. The glucosyl backbone starts to emerge from the binding cleft from site +2. None of the sugar residues at sites +2, +3, and +4 makes direct H-bonding contacts with protein atoms but are found to be involved in a complex network of interactions mediated by water molecules as is represented in Fig. 6B. In the Xgh74A complex described here there is no density, not even at very low levels, indicative of a galactosyl residue attached to the +2' xyloside. Indeed, inspection of the structure would suggest that there are steric blockages for the accommodation of a +2 "galactosyl moiety. The interpretation of limit digest patterns, in particular the observation of XLLG, demands that the +2" region must be able to accommodate a galactosyl moiety during catalysis. One possibility is that the binding mode for XLLG is more flexible, at either protein or ligand levels, than that observed here for XXLG.
Conservation of Xyloglucan Recognition Sites in Members of GH74 FamilyGH74 family groups enzymes that are able to hydrolyze xyloglucan oligosaccharides but also are active on non-branched substrates like barley The specificity of the interaction between xylo-oligosaccharide ligands and Xgh74A interpreted in light of direct H-bonding contacts with the enzyme is reduced to positions -3', -2', -2, -1, +1, and +1' (Fig. 8). Apart from the strict conservation of the catalytic residues Asp70 and Asp480 and their sequence equivalents in the GH74 family, the residues responsible for the recognition of the glucosyl units at positions -2, -1, and +1 appear highly conserved in the multiple sequence alignment of GH74 members (Fig. 8). Two substitutions are only observed at the glucosyl recognition site -1 and +1 in the multiple sequence alignment. The first one is in the equivalent position to Xgh74A Phe51 where a Tyr residue appears in Geotrichum sp. OXG-RCBH and in Aspergillus nidulans OREX. This interaction is not mediated by the side chain but through an H-bond between the carbonyl oxygen of the main chain and the Glu-1 O1 atom. The second substitution is observed in the equivalent position of Xgh74A Trp410 where a His residue appears in T. maritima endoglucanase. The sequence around this region in T. maritima Cel74 is also different, a Pro residue appears in the position of an otherwise strictly conserve Gly residue in a two residues shorter loop. The sequence equivalent position of Xgh74A Asn749 responsible for the interaction with the O2 and O3 of glucosyl residue at position -2 appears also highly conserved in the GH74 alignment.
The equivalent positions of Xgh74 residues responsible for xyloglucan recognition at the prime subsites are not as well conserved at the sequence level as the positions at the -/+ 1 and -2 subsites. The degree of sequence variation increases from position -2' where some conservation among the GH74 members is observed to position -3' where no conservation is observed among the family GH74 members. Thus, the equivalent to Xgh74 Tyr295 that recognizes a xylose residue at the -2' subsite appears conserved in most of the sequences with the exception of the endoglucanase from T. maritima where an Asn residue is found instead. The equivalent position of Xgh74 Asp524 that contacts the xylose residue at the -1' subsite appears as Ile, Val, or Ala in the xyloglucanases from Jonesia sp., Thermobifida fusca and Hypocrea jecorina respectively. At the -3' subsite, Xgh74 Asp731 is found not conserved in any of the family members, the loop in which this residue is located displays variations in length and overall amino acid composition making difficult to assess the presence or not of an equivalent interaction in the absence of structural data on these other family members. Exo versus Endo Specificity in Family GH74In both Xgh74A and OXG-RCBH the substrate binding cavities are open grooves well exposed to solvent (Fig. 9). OXG-RCBH is an exoglucanase that releases two glucosyl residues from the reducing end of the xyloglucan polymer, suggesting the presence of at least two negative reducing end subsites and two positive leaving group subsites (22, 24) and demanding the ability to cleave at xylose-substituted glucosyl moieties. In contrast, Xgh74A processes the xyloglucan chain in an endo fashion releasing four glucosyl residue segments (Ref. 19 and this work). Xgh74A residues Trp125 and Asp731 appear to contribute to specificity for the sugars located at subsite -3 where they interact directly with Glc-3 and Xyl-3' (Figs. 5 and 6). In the OXG-RCBH structure, the equivalent structural determinants for sugar recognition at the -3 site are absent. The OXG-RCBH equivalent to Trp125 of Xgh74A is Asp89, which is situated in a loop two residues shorter than in Xgh74A and is consequently too distant to interact with the Glc-3 residue. Similarly, while Xgh74A Asp731 interacts directly Xyl-3' O2 and O3 atoms, the structural equivalent of Xgh74 Asp731 in OXG-RCBH is Thr736, but its position is again distant from Xyl-3' most likely as a result of conformational constraints imposed in the loop by the presence of residues Gly734-Pro735 in OXG-RCBH. These features likely contribute to the reported differences between the two enzymes with respect to the number of reducing end subsites. Differences between the two enzymes at the +3 subsite (Fig. 9) are more pronounced. The conformations of the (structurally equivalent) loops Xgh74A Thr397-Pro406 and OXG-RCBH Asn374-Thr391 are dramatically different; the latter closing the binding cleft immediately after the subsite +2 and presumably restricting OXG-RCBH to exo-hydrolysis. The cleft in Xgh74A is thus open in both extremes and differs from OXG-RCBH in which the loop Gly375-His385 blocks one-half of the substrate binding landscape. The Xgh74A structure reveals both a complex binding architecture in which subsites accommodate seventeen distinct sugar moieties accounting for the pattern of xyloglucan recognition observed by limit hydrolysis of tamarind xyloglucan. Catalysis occurs with inversion of anomeric configuration in a mechanism in which (kinetically essential) aspartates 480 and 70 likely play the role of catalytic acid and base, respectively. Given the growing importance of plant biomass conversion, especially in the context of the demand for clean energy sources, the Xgh74A structure provides the first insights into the recognition and hydrolysis of this crucial component of the plant cell wall.
The atomic coordinates and structure factors (codes 2CN2 and 2CN3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
* This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the Swedish Foundation for Strategic Research, the KTH Biofibre Materials Centre, and the Fundação para a Ciência e Tecnologia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 These authors contributed equally to this work.
2 Fellow (Rådsforskare) of the Swedish Research Council. 3 To whom correspondence should be addressed. E-mail: davies{at}ysbl.york.ac.uk.
4 The abbreviations used are: Ct, Clostridium thermocellum; RMSD, root mean-squared deviation; PDB, Protein Data Bank; PEG, polyethyleneglycol; HPAEC-PAD, high performance anion-exchange chromatography with pulsed amperometric detection; CAZy, carbohydrate active enzyme; GH, glycoside hydrolase; XGO, xylogluco-oligosaccharides; CMC, carboxymethyl cellulose.
We thank Gustav Sundqvist (KTH Biotechnology) for MS analysis.
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