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Originally published In Press as doi:10.1074/jbc.M603583200 on June 13, 2006

J. Biol. Chem., Vol. 281, Issue 34, 24922-24933, August 25, 2006
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Crystal Structures of Clostridium thermocellum Xyloglucanase, XGH74A, Reveal the Structural Basis for Xyloglucan Recognition and Degradation*

Carlos Martinez-Fleites{ddagger}1, Catarina I. P. D. Guerreiro§1, Martin J. Baumann1, Edward J. Taylor{ddagger}, José A. M. Prates§, Luís M. A. Ferreira§, Carlos M. G. A. Fontes§, Harry Brumer2, and Gideon J. Davies{ddagger}3

From the {ddagger}York Structural Biology Laboratory, Department of Chemitry, University of York, York YO10 5YW, United Kingdom, the §CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal, and the School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden

Received for publication, April 13, 2006 , and in revised form, June 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 beta-1,4 D-glucan regularly substituted with {alpha}-1,6 D-xylosyl units, which is, in a species-dependent manner, further derivatized with {alpha}-L-arabinose or beta-D-galactose (2, 3), Fig. 1. In primary cell wall xyloglucans, the first galactose moiety in the oligosaccharide repeat is commonly substituted with {alpha}-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 beta-glucanase (lichenase) enzymatic activity (16). Cellulosome enzymes are tethered to the scaffolding protein of the complex through the interaction of dockerin domains with one of the nine cohesin platforms of the scaffold (17), Fig. 2A.

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-beta-xylanase and a xyloglucanase (19). Xyloglucanase Xgh74A, Fig. 2B, is the first xyloglucanase identified in Ct and the first active xyloglucanase in a cellulosome and likely plays an important role in the degradation of dicot plant cell wall polysaccharides.


Figure 1
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  		FIGURE 1.
The general structure of xyloglucan. Endoglucanases, including Xgh74, typically cleave the glycosidic bond of the unbranched Glc residue (arrow) to yield xylogluco-oligosaccharides. Common structures in seed xyloglucans (e.g. Tamarindus indica) include XXXG (x = 0, y = 0, R = H), XLXG (x = 1, y = 0, R = H), XXLG (x = 0, y = 1, R = H), and XLLG (x = 1, y = 1, R = H). XXFG (x = 0, y = 1, R = {alpha}-1,2 L-Fuc) is found in xyloglucans from dicot primary cell walls. Oligosaccharide nomenclature is according to Ref. 23.

 
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 beta-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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
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
Mutants of Xgh74A were generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions and using pCG1 as the template DNA. The sequences of the primers used to generate the protein mutants were as follows: 5'-GATTTATGCACGTGCCGCTATCGGAGGAGCGTACC-3' and 5'-GGTACGCTCCTCCGATAGCGGCACGTGCATAAATC-3', D70A; 5'-CTTGTAAGTGCAGTTGGGGCCCTTGTCGGTTTTGTTC-3' and 5'-GAACAAAACCGACAAGGGCCCCAACTGCACTTACAAG-3', D480A. The mutated DNA sequences were sequenced to ensure that only the appropriate mutations had been incorporated into the nucleic acid.

Production of Recombinant Xgh74A and Mutants
E. coli Tuner cells (Novagen) harboring the pET21a-Xgh74A plasmid were cultured in LB containing ampicillin to mid-exponential phase (A600 0.6) at which point, cultures were transferred to 20 °C and induced by addition of 1 mM isopropyl 1-thio-beta-D-galactopyranoside (IPTG) whereupon they were grown for a further 20 h. SeMet-labeled Xgh74A was produced in E. coli B834 (DE3) containing the pET21a-Xgh74A plasmid with recombinant protein expression induced by 1 mM IPTG and incubation at 20 °C for 20 h. Cells were harvested by centrifugation and disrupted by sonication in 20 mM HEPES-NaOH, 400 mM NaCl, pH 7.5 buffer. The cell-free extract was incubated at 65 °C for 15 min and centrifuged to remove insoluble material. Samples were further purified by Ni2+ affinity chromatography and buffer exchanged to 10 mM HEPES-NaOH pH 7.5. Xgh74A samples thus purified were assessed pure by SDS-PAGE and were used for crystallization experiments. Xgh74A-D70A and D480A mutants were produced and purified following the native Xgh74A expression and purification protocols.

High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)
Oligosaccharides were analyzed on a Waters HPLC system with a Dionex Carbopac PA100 column. A Waters Concorde electrochemical detector was used in PAD mode with a 3-mm gold electrode and a HyREF platinum reference electrode. Two optimized gradients were used for different sizes of xylogluco-oligosaccharides.

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
Mass spectrometric analysis was performed with a Q-T ofTM 2 mass spectrometer fitted with a nanoflow ion source (Waters Corporation, Micromass MS Technologies, Manchester, United Kingdom). External calibration of the TOF analyzer (single-reflectron mode, resolution >10000 FWHM) was obtained over the m/z range 50-1000 using a solution of NaI (1.5 g/liter) in 1:1 2-propyl alcohol/water. Solutions of xylogluco-oligosaccharides (typical concentration 0.01-0.1 g/liter in 1:1 MeOH/water containing 0.5 mM NaCl) were infused into the ion source (3 kV) at 200 nl/min (syringe pump). The cone voltage was varied between at 35 V and 130 V to optimize the intensity of [M+Na]+ and [M+2Na]2+ ions. Argon was present in the collision cell at all times, and the collision energy was 10 V. A scan time of 2.5 s with an interscan delay of 0.1 s was used, and continuum data were collected until an acceptable signal-to-noise ratio was achieved after the combination of individual spectra (typically 1-30 spectra).

Preparation of Xylogluco-oligosaccharides (XGOs) from Deoiled Tamarind Kernel Powder
Mixture of Xylogluco-oligosaccharides Based on a Glc4 Backbone (XLLG, XLXG, XXLG, XXXG)—Deoiled tamarind kernel powder (Saiguru Food Gum Manufacturer, Mumbai, India) (20 g) was suspended in ammonium acetate buffer (1 liter; 10 mM; pH 4.5) at 60 °C and was vigorously stirred until homogenous. Then the suspension was cooled to 30 °C and crude cellulase from Trichoderma reesi (Fluka) was added (100 mg, 500 units). The resulting solution was incubated at 30 °C during 18 h under gentle stirring. The progress of the digestion was monitored by HPAEC-PAD (gradient B). The solution was filtered on a glass fiber filter, 1 ml of NH3 (37% in H2O) was added, and the basic solution was pumped over a Q-Sepharose (GE Healthcare) column (10 cm high, 2.6 cm diameter) to remove the cellulase. The resulting solution was freeze-dried to yield a mixture of XGOs as a white powder (typical yield 8-9 g). The following oligosaccharide composition was obtained by HPAEC-PAD (gradient A): XXXG, XLXG, XXLG, XLLG (2:1:3:3). Mixture of Higher Order Xyloglucan Oligosaccharides (Glc8-Glc16 Backbone)—A modified protocol based on that described by Vincken et al. (26) was used. One gram of deoiled tamarind kernel powder was dissolved in ammonium acetate buffer (50 ml; 10 mM; pH 4.5) at 60 °C for 1 h. The solution was cooled to 30 °C and 1 mg (1 unit) of crude cellulase (T. reseei, Fluka) was added. The progress of the digestion was monitored by HPAEC-PAD (gradient B). When HPAEC-PAD analysis indicated the presence of predominantly Glc4 and Glc8 oligosaccharides, the digestion was stopped (16 h) by boiling for 30 min. The solution was cooled to room temperature, filtered on a glass fiber filter, and the filtrate concentrated in vacuo to a volume of 10 ml. The oligosaccharides were separated by size exclusion chromatography on two Bio-Gel P6 (Bio-Rad) columns (2 x 90 cm, 2.6 cm diameter) connected in series and maintained at 60 °C. The products were eluted with a flow rate of 0.5 ml/min with ultrapure water. Fractions (5 ml) were analyzed by HPAEC-PAD (gradient B), pooled; concentrated in vacuo, and finally freeze-dried (typical yield 130 mg of Glc4 oligosaccharides, 190 mg of Glc8 oligosaccharides, 98 mg of Glc12 oligosaccharides, 20 mg of Glc16 oligosaccharides and 8 mg of Glc20 oligosaccharides).

XXXGXXXG and Partially Degalactosylated Glc12-based Xyloglucan Oligosaccharides—Degalactosylated higher order oligosaccharides were produced exactly as described in the preceding paragraph, except that 4 units of beta-galactosidase (Aspergillus niger, Megazyme, Eire) were added immediately after boiling and cooling the crude cellulase digestion mixture. The degalactosylation reaction was carefully controlled by HPAEC-PAD analysis (gradient B) because of contaminating isoprimeverase activity in the commercial beta-galactosidase (27).

Crystallization, Data Collection, Structure Solution, and Refinement
Crystals of SeMet-Xgh74A and Xgh74A-D70A in complex with xyloglucan-derived oligosaccharides were grown by the hanging-drop method. SeMet-Xgh74A (10 mg/ml) was crystallized in 5 mM CdCl2, 12% PEG 4000, 100 mM sodium cacodylate, pH 6.5. Xgh74A-D70A-XXLG complex crystals were prepared by co-crystallization of the Glc4 XGO mixture and the inactive mutant (46 mg/ml) in 200 mM KSCN, 22% PEG 3350, 100 mM HEPES-NaOH, pH 7.5. In both cases, crystals were cryoprotected by the addition of 20% glycerol (v/v) to the crystallization conditions. A 2.1-Å resolution dataset was collected from an uncomplexed SeMet-Xgh74A crystal at 100 K on beamline ID14.3 (ESRF). Diffraction data of the Xgh74A-D70A-XXLG complex were collected on beamline ID23.1 (ESRF). Both data sets were integrated with MOSFLM and scaled and reduced with SCALA from the CCP4 suite of programs (28). Crystallographic statistics for data collection are summarized in Table 2.


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  		TABLE 2
Data collection and refinement statistics

 


Figure 2
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  		FIGURE 2.
Schematic of the cellulosome (A) and of xyloglucanase Xgh74A (B).

 
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
Kinetics of Xgh74A—Reducing sugars released from the hydrolysis of plant cell wall polysaccharides by Xgh74A were quantified following the method described by Nelson (33) and Somogyi (32). Enzyme activities were measured in 25 mM potassium phosphate buffer, pH 7.0, at 50 °C. The assay mixture (100 µl) contained 10 µl of enzyme (with appropriate dilution) and concentrations of substrate ranging from 0.05 to 15 g/liter. Activities were determined in triplicate in the linear range of the reactions.

Limit Digest of Tamarind Xyloglucan and Glc4-, Glc8-, and Glc12-based Xylogluco-oligosaccharides by Xgh74A—Tamarind 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.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
The Ct beta-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 Xgh74A—Xgh74A 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.


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  		TABLE 1
Kinetics of Xgh74A on different polysaccharides

 


Figure 3
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  		FIGURE 3.
HPAEC-PAD analysis (gradient A) of endoglucanase hydrolysis products. A, Xgh74A limit digest of tamarind xyloglucan. B, Trichoderma EGII limit digest of tamarind xyloglucan. C, Xgh74A limit digest of partially degalactosylated Glc12-based xyloglucan-oligosaccharides. D, Trichoderma EGII limit digest of partially degalactosylated Glc12-based xyloglucan-oligosaccharides.

 
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 beta (1-4) glycosidic bond between two branched Glc units bearing {alpha} (1-6) Xyl residues has been previously reported in the related GH74 oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH) from Geotrichum sp (24). Incubation of a high concentration of Xgh74A with a 2:1: 3:3 mixture of XXXG/XLXG/XXLG/XLLG showed no formation of smaller oligosaccharides by HPAEC-PAD (data not shown) demonstrating that Xgh74A, in contrast to OXG-RCBH, cannot cut at substituted glucosyl moieties, at least on these short substrates. Similarly, incubation of Xgh74A with XXXGXXXG or a variably galactosylated Glc8 XGO mixture, yielded only XXXG or the expected XXXG/XLXG/XXLG/XLLG mixture, respectively, as determined by HPAEC-PAD (data not shown). Likewise, Xgh74A digestion of a partially degalactosylated Glc12 XGO mixture yielded predominantly XXXG and minor amounts of other Glc4-based XGOs (Fig. 3). In all cases the action of Xgh74A, as determined by HPAEC-PAD and/or MS analysis, was indistinguishable from that of commercially available T. longibrachiatum endoglucanase (Fig. 3).

Structure of C. thermocellum Xyloglucanase Xgh74A—The 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).


Figure 4
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  		FIGURE 4.
Mass spectrometric analysis of the hydrolysis products of Xgh74A acting on tamarind xyloglucan. A, cone voltage 35 V. B, cone voltage 130 V.

 
Xgh74A consists of two sevenbladed beta-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).


Figure 5
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  		FIGURE 5.
Three-dimensional structure of C. thermocellum xyloglucanase Xgh74A. A, divergent (wall-eyed) stereo cartoon of the structure, color-ramped from N terminus (blue) to C terminus (red) with the ligands in ball-and-stick representation. B, mono view of the structure showing the N- and C-terminal domains, presumably resulting from an ancestral gene duplication event, colored khaki and pale blue, respectively. This figure was drawn with MOLSCRIPT (40).

 
The overall topology of Xgh74A is thus very similar to OXG-RCBH with all the secondary structure elements linked through identical connectivity. The C{alpha} 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 beta-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 Structure—It 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 beta-propeller blades in both domains. In the apo Xgh74A structure some of these loops (Tyr206-Asp217, Thr291-Asn298, and Asp524-Asp527) are disordered, whereas in the ligand complexed forms they become ordered and participate in substrate binding (described below).

Catalysis by family GH74 enzymes occurs with inversion of anomeric configuration; i.e. the stereochemistry of the product is inverted with respect to the beta-linkage of the substrate. A classical interpretation of glycoside hydrolysis with inversion of anomeric configuration implicates two key residues; a catalytic acid, to facilitate leaving group departure by protonation and a catalytic base, to activate the incoming water molecule for nucleophilic attack by deprotonation (glycosidase catalytic mechanisms are reviewed in Ref. 25). In GH74 enzymes, it is believed (22) that two aspartate residues play the role of Brønsted acid and base; in Xgh74A these are believed to be Asp480 and Asp70, respectively. Asp70 and Asp480 are located in the middle of the active center cleft, lying on opposite sides, and deep within the cavity with their carboxylate groups ~10 Å apart. Site-directed mutagenesis of either of these residues, to alanine, results in an inactive enzyme (which within the sensitivity of the assay suggests at least 1000-5000 times less activity that wild-type enzyme). Asp70 is located in the middle of the loop connecting the second and third strand of the first propeller blade of the N-terminal domain. The peptide sequence in this region is strictly conserved among the members of the GH74 family. In the apoenzyme, Asp70 forms a hydrogen bonding interaction with the side chain of Glu459 and two molecules of water. Asp480 is located in the C-terminal domain in an equivalent position to Asp70. However, in contrast to Asp70, Asp480 does not form H-bonds with protein atoms and points directly into the cleft. It is not immediately apparent what might contribute to an elevated pKa for this catalytic acid in the absence of bound substrate.


Figure 6
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  		FIGURE 6.
Interactions of C. thermocellum xyloglucanase Xgh74A (D70A) with two xyloglucan-derived oligosaccharides. A, observed 2Fobs-Fcalc electron density for the ligand with interacting residues (drawn with BOBSCRIPT, Ref. 41). "Negative" subsite sugars are shown in cyan with "positive" (leaving group) subsite sugars in yellow. Residues making direct interactions with the ligands are shown in gray. B, ligand binding to Xgh74A (D70A) in this case, solvent-mediated hydrogen bonds are included with their protein partners colored in blue and with the residues making direct contacts remaining in gray.

 
Structure of the Xgh74A-D70A Mutant in Complex with Glc4-based Xylogluco-oligosaccharides—To 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{alpha} 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 ~316 Å2.

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 beta-1,4 glucosyl moieties of one XLLG molecule are located in the negative binding sites-1 (Glc-1), -2 (Glc-2), -3 (Glc-3), and -4 (Glc-4) in an extended conformation. Three {alpha}-1,6-linked xylose residues branch from the -2 (Xyl-2'), -3 (Xyl-3'), and -4 (Xyl-4') glucosyl units, with both Xyl-2' and Xyl-3' also bearing a beta-1,2-linked galactosyl (Gal-2'') unit, the latter partially disordered. The mean temperature factor for this oligosaccharide is 16 Å2 and its interaction area with the enzyme is ~162 Å2. Glc-1 is positioned in the middle of the diagonal line connecting the C{alpha} atoms of the catalytic residues Asp70 and Asp480 (Ala70 in the complex structure). Ala70 in the Xgh74A-D70A mutant lies below the plane of the Glc-1 ring at a distance of about 5.8Å between Glc-1 C1 and Ala70 C{alpha}, consistent with the position demanded for the catalytic base in an inverting mechanism. Asp480 is located above this plane at a distance of 4.3 Å. Glc-1 forms a network of H-bonding interactions where all its oxygen atoms except O4 are involved (Figs. 6 and 7). Glc-1 O1, O2, and O3 atoms interact with the nitrogen main chain of Phe51, Arg158 side chain and Asn154 side chain respectively, whereas the putative catalytic acid, Asp480, forms H-bonds with Glc-1 O5 and O6. All the side chains involve in the recognition of Glc-1 appear conserved in the multiple sequence alignment of GH74 family, Fig. 8. Superposition of the Xgh74A and OXG-RCBH structures shows similar environments around the Glc-1 O6 position (-1' subsite). In both structures, the loops enclosing this region adopt similar conformations and some of the side chains lining the cavity are conserved, leaving room to accommodate a xylose residue at the -1' position. Accordingly, it is not evident what constitutes the structural basis supporting the fact that endoxyloglucanases prefer to cleave the xyloglucan chain at unbranched glucosyl positions (-1) in contrast with the ability of exo-xyloglucanases to process substrates with a xylose ramification at the -1' position (24, 35).


Figure 7
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  		FIGURE 7.
Schematic diagram of the direct interactions of Xgh74A (D70A mutant) with two xyloglucan-derived oligosaccharides.

 
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 ~90 degrees with respect to the plane of Glc-3 ring pointing directly to the middle of the cleft in the N-terminal to C-terminal direction. This residue forms two H-bonds between its O3 and O4 atoms and the side chain of Asp731. An additional partially occupied Gal (Gal-3'') residue was modeled in the electron density adjacent to Xyl-3' O2 atom. This Gal-3'' residue is located at the entrance of the cleft and exposed to solvent. Gal-3'' does not interact directly with any protein atom but forms one H-bond between its O4 and the O3 of Glc-3. This structural feature is in agreement with the described absence of specificity toward Gal residues on positions -2 and -3. The sugar moieties at position -4, Glc-4 and Xyl-4' are located at the periphery of the cleft. The plane of the Glc-4 ring lies at an angle of 180 degrees with respect to the plane of Glc-3. This glucosyl unit contacts the enzyme through an H-bond between O2 and main chain oxygen of Trp125. The Xyl-4' residue does not contact any protein atom and only makes H-bonding interactions between its O4 atom and Gal-2' O4 and Glc-2 O2 atoms.


Figure 8
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  		FIGURE 8.
Sequence conservation around the xyloglucan oligosaccharide recognition sites in family GH74. A, schematic representation of the sugar subsites specifically recognized with Xgh74A (bold circles). B, fragments of the multiple sequence alignment of members of family GH74 whose enzymatic characterization has been reported. The sequence alignment was calculated with ClustalW (42) and represented with ESpript (43). Psp, Paenibacillus sp. KM21; Jsp, Jonesia sp. DSM 14140; Tf, Thermobifida fusca YZ, Aa, Aspergillus aculeatus; Anig, A. niger; Hj, Hypocrea jecorina QM6a; Tm, T. maritima; Gsp, Geotrichum sp. M128; Anid, A. nidulans FGSC A4.

 
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 Family—GH74 family groups enzymes that are able to hydrolyze xyloglucan oligosaccharides but also are active on non-branched substrates like barley beta-glucans (beta-1,3/1,4 glucan), carboxymethyl cellulose (CMC), Avicel (microcrystalline cellulose) or galactomannan (beta-1,4-mannose). A wide spectrum of activities is observed among members of this family. This spectrum covers enzymes that can, apparently, only process xyloglucan up to enzymes that actually prefer non-branched substrates such as barley beta-glucan. For example, Paenibacillus sp. KM21 (36) and Thermobifida fusca YZ (37) xyloglucanases are only active on xyloglucans from various sources but not on nonbranched polymers such as barley beta-glucan, CMC, Avicel, or xylan. Geotrichum sp. M128 xyloglucanase displays its highest activity on the more branched xyloglucans from tamarind or pea, but it is less efficient on barley xyloglucan that contains fewer xylose decorations and the enzyme shows no activity on non-branched substrates (44). At the other end of the spectrum, Thermotoga maritima Cel74 shows its highest activity on barley beta-glucan and is about 75% less efficient on tamarind xyloglucan (38).

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.


Figure 9
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  		FIGURE 9.
Comparison of endoxyloglucanase Xgh74A and the reducing end-specific GH74 enzyme OXG-RCBH. A, van der Waals' surface for Xgh74A (yellow) with the ligands in blue showing the open cleft of this endoenzyme. B, van der Waals' surface for OXG-RCBH (pale green) with the ligands from Xgh74A superimposed in yellow ball-and-stick. The surface of the OXG-RCBH loop Gly375-His385 that closes off the substrate binding surface and is responsible for the exo mode of action is shaded in red. The figures are shown in divergent ("wall-eyed") stereo.

 
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 GH74—In 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.


   FOOTNOTES
 
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. Back

1 These authors contributed equally to this work. Back

2 Fellow (Rådsforskare) of the Swedish Research Council. Back

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. Back


   ACKNOWLEDGMENTS
 
We thank Gustav Sundqvist (KTH Biotechnology) for MS analysis.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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