The crystal structure of a xyloglucan-specific endo- b -1,4- glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity Katsuro Yaoi 1, *, Hidemasa Kondo 2, *, Ayako Hiyoshi 1 , Natsuko Noro 2 , Hiroshi Sugimoto 3 , Sakae Tsuda 2,4 and Kentaro Miyazaki 1,5 1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan 2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira, Sapporo, Hokkaido, Japan 3 Riken SPring-8 Center, Harima Institute, Hyogo, Japan 4 Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Japan 5 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tsukuba, Ibaraki, Japan Introduction Xyloglucan is a major hemicellulose in the primary cell wall of plants, where it associates with cellulose micro- fibrils via hydrogen bonds to form a cellulose–xyloglu- can network. Xyloglucan consists of a cellulose-like main chain of b-1,4-glucan, which is frequently branched with xylose side chains to form a-d-Xylp- Keywords endo-b-1,4-glucanase; glycoside hydrolase family 74; xyloglucan; xyloglucanase; b-1,4-glucan Correspondence K. Yaoi, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Fax: +81 29 861 6733 Tel: +81 29 861 6065 E-mail: k-yaoi@aist.go.jp *These authors contributed equally to this study Database The coordinates of the structure for XEG have been deposited in the Protein Data Bank under the accession number 3A0F (Received 14 May 2009, revised 3 July 2009, accepted 8 July 2009) doi:10.1111/j.1742-4658.2009.07205.x Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-b-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct. XEG catalyzes the hydrolysis of xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on xyloglucan oligosaccharides at the reducing end in exo mode. Here, we determined the crystal structure of XEG at 2.5 A ˚ resolution, and compared it to a previously determined structure of OXG-RCBH. For the most part, the amino acid residues that interact with substrate are conserved between the two enzymes. However, there are notable differences at subsite posi- tions )1 and +2. OXG-RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action. In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the substrate. At the )1 site in OXG-RCBH, Asn488 interacts with the xylose side chain of the substrate, whereas the )1 site is occupied by Tyr457 in XEG. To confirm the contri- bution of this residue to substrate specificity, Tyr457 was substituted by Gly in XEG. The wild-type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same substrate, but at various sites. Together, the absence of a loop in the cleft and the presence of bulky Tyr457 determine the substrate specificity of XEG. Abbreviations GH74, glycoside hydrolase family 74; Glc, glucose; OXG-RCBH, oligoxyloglucan reducing-end-specific cellobiohydrolase; XEG, xyloglucan- specific endo-b-1,4-glucanase; Xyl, xylose. 5094 FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS (1 fi 6)-b-d-Glcp. Other sugars such as galactose, arabinose and fucose may also be present on the side chains in various branching patterns, depending on the plant species. Structural studies on xyloglucans suggest that most consist of repeating units of either XXXG (XXXG-type) or XXGG (XXGG-type) [1], where G refers to an unbranched Glc residue and X represents the xylose (Xyl)-branched Glc a-d-Xyl p-(1 fi 6)-b-d- Glcp [2]. Thus, XXXG-type xyloglucans have three consecutive backbone residues with Xyl side chains and a fourth unbranched Glc residue, and XXGG-type xyloglucans have two consecutive branched backbone residues and two unbranched backbone residues. Various microorganisms that degrade the plant cell wall produce endo-b-1,4-glucanases that hydrolyze xyloglucan. Most of them cleave the glycosidic bond of the unbranched Glc residues in the backbone chain. Typically, treatment of an XXXG-type xyloglucan with these endoglucanases generates oligosaccharides with a tetrasaccharide backbone (XXXG). For many years, endo-b-1,4-glucanases have been considered to be a subgroup of cellulases (EC 3.2.1.4). Recently, however, it has been clarified that some endo-b-1,4- glucanases display high activity toward xyloglucan but have limited or no activity against cellulose. These xyloglucan-specific endo-b-1,4-glucanases have been classified as a new enzyme family (EC 3.2.1.151), desig- nated xyloglucan-specific endo-b-1,4-glucanases (XEG) or xyloglucanases. Many xyloglucanases belonging to glycoside hydrolase families 5, 12, 44 and 74 (http:// www.cazy.org/) have now been identified. Of these, glycoside hydrolase family 74 (GH74) enzymes exhibit high specificity towards xyloglucan [3–9]. Previously, we have reported the cloning, purifica- tion and characterization of two GH74 enzymes, oligo- xyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) [6] and xyloglucan-specific endo-b-1,4- glucanases (XEG) [7], from Geotrichum sp. M128. OXG-RCBH (EC3.2.1.150) is a unique exo-type enzyme that recognizes the reducing end of xyloglucan oligosaccharides. Its substrate recognition mechanism has been investigated using various oligosaccharide substrates [6], and mutational and detailed structural studies have revealed its unique mechanism [10,11]. The exo activity of OXG-RCBH is based on a loop at one side of the active site cleft. In addition, residue Asn488 in the active site cleft recognizes the Xyl side chain at the )1 site, conferring the unique substrate specificity. On the other hand, XEG showed typical endo activity [12]. It hydrolyzes xyloglucan polymers randomly. In addition, XEG catalyzes hydrolysis of the glycosidic bond of unbranched Glc residues, sug- gesting a difference between the two enzymes in the three-dimensional structures of the active sites. In this study, we determined the crystal structure of XEG in order to determine the structural basis for its substrate specificity. Results and discussion Overall structure of XEG Crystals were grown using 0.1 m MES buffer, pH 5.8– 6.0, and 3–6% w ⁄ v PEG 8000. They were elongated rod- or needle-shaped crystals, belonging to the P3 2 21 trigonal space group, with unit-cell parameters of a = b = 135.2 A ˚ and c = 119.9 A ˚ . One protein molecule of XEG existed in an asymmetric unit of the crystal. X-ray diffraction data were collected at 2.5 A ˚ resolution, with an R merge of 0.096 and 98.4% com- pleteness. The structure was determined using the molecular replacement method and was refined against 20–2.5 A ˚ intensity data. The crystallographic R factor and free R factor were 0.236 and 0.276, respectively. Table 1 summarizes the statistics of X-ray data collec- tion, and the results for the structural refinement of Table 1. Statistics for data collection using the Beamline BL44B2 at SPring-8 and structure refinement of XEG. Parameter Value Data collection Wavelength (A ˚ ) 1.0000 Resolution a (A ˚ ) 20–2.5 (2.59–2.5) R merge a,b 0.096 (0.199) Observed reflections 267 842 Independent reflections 43 425 Completeness a (%) 98.4 (93.2) Multiplicity a 6.2 (6.0) <I ⁄ r(I)> a 17.9 (8.6) Refinements Resolution range a (A ˚ ) 20–2.5 (2.56–2.5) R factor a,c 0.236 (0.251) Free R factor a,c 0.276 (0.296) Number of non-hydrogen atoms Protein 5 676 Water 107 Root mean square deviations from ideality (bond length) (A ˚ ) 0.015 Root mean square deviations from ideality (angle) (°) 1.64 a Numbers in parentheses are values for the highest-resolution shell: 2.59–2.5 A ˚ for the data collection and 2.56–2.5 A ˚ for the refinement. b R merge ¼ P h P j <Iðh Þ> À I j ðh Þ     = P h P j I j ðhÞ, where <I(h)> is the mean intensity of a set of equivalent reflections. c R factor ¼ P h F obs ðhÞÀF calc ðhÞ jj = P h F obs ðh Þ jj , where F obs and F calc are the observed and calculated structural factors, respectively. K. Yaoi et al. Key amino acid residue of Geotrichum XEG FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS 5095 XEG. Figure 1 shows the electron density map of XEG for the region corresponding to the exo-loop of OXG-RCBH. The coordinates and structure factors have been deposited in the Protein Data Bank (PDB) (accession code 3A0F). The structure of XEG was compared with those of other GH74 xyloglucan-specific enzymes, OXG-RCBH and Clostridium Xgh74A. The overall structures of XEG (this study), OXG-RCBH in complex with substrate XXXG (PDB code 2EBS) and Xgh74A in complex with substrates XLLG and XXLG (L refers to a b-d-Galp-(1 fi 2)- a-d-Xylp-(1 fi 6)-b-d-Glcp) (PDB code 2CN3) [13] are illustrated in Fig. 2. All enzymes have two structurally similar domains, each consisting of a seven-bladed b-propeller. These are located tandemly in the N- and C-terminal halves of the poly- peptide, and are joined on each edge of the domain to form a bivalve-like shape. XEG can be superimposed onto OXG-RCBH with an RMSD of 1.07 A ˚ by the corresponding 683 Ca atoms among 756 residues, indi- cating close similarities between their domain structures as well as the relative positions of the two domains. The active site cleft is located between the N- and C-domains. One apparent difference between XEG and OXG-RCBH is at the active site. In OXG-RCBH, a loop comprising 11 amino acid residues protrudes from the N-domain, blocking one end of the cleft. However, XEG lacks a corresponding loop because of deletion of those residues. The loop structure is responsible for the exo-activity of OXG-RCBH, and the absence of the loop is associated with endo-activity in XEG. Therefore, it is most likely that the endo- activity of XEG is primarily attributable to the active site cleft being open at both ends. In the case of Xgh74A, the active cleft is open. Although a precise anal- ysis of the mode of action of Xgh74A has not been performed, Xgh74A appears to be an endoglucanase because it has an open cleft. Comparison of the XEG and OXG-RCBH active sites Figure 3 shows a close-up view of the active sites of XEG and OXG-RCBH. The main chain of XEG exhibits close similarity to that of OXG-RCBH, and the side chain conformations involved in substrate interactions are very well conserved, with some excep- tions (see below for details). Previously, we have iden- tified the catalytic residues Asp35 (base) and Asp465 (acid) in OXG-RCBH [10]. Equivalent residues were identified in the active site of XEG (Asp34 and Asp458). Thus, it is conceivable that XEG and OXG-RCBH recognize the backbone of the b-1,4-glu- can in a similar manner, despite their differences in substrate specificity and mode of hydrolysis. Two notable differences, located at the )1 and +2 sites, were observed. OXG-RCBH functions in exo mode owing to its unique loop structure around the +2 site at one end of the cleft (Figs 2B and 3, shown in red). The Xyl side chain at the +2 site inhibits enzymatic activity due to steric hindrance between the side chain and the exo-loop. Previously, we demon- strated that deletion of the loop region leads to loss of specificity, as the resultant enzyme could catalyze cleavage at various sites of the substrate by endo activ- ity [10]. XEG does not have a corresponding loop; both sides of the cleft are open. This result, and those of the previous experiment with loop-deleted OXG- RCBH [10], strongly suggest that the basis for the endo activity of XEG is the absence of the exo loop. The second difference was observed at the )1 site. In OXG-RCBH, Asn488 interacts with a Xyl side chain at the )1 site. Previously, we have shown that recognition of the Xyl side chain at the )1 site is a key determinant for the substrate specificity of OXG- RCBH [6]. However, the corresponding position in XEG is occupied by the bulky side chain of Tyr457, which appears to hinder binding of the Xyl side chain at this position. This may explain why XEG prefers Fig. 1. Stereoview of the r A -weighted 2m|F obs | ) D|F calc | map of XEG at 2.5 A ˚ resolution, contoured at 1.5r. The map shows the vicinity of the region corresponding to the exo loop, which is pres- ent in RCBH and absent in XEG. The XEG molecule is also shown as a stick model. The exo loop of RCBH is drawn as a trace of Ca atoms in orange. The image was produced using the program CCP4MG [22]. Key amino acid residue of Geotrichum XEG K. Yaoi et al. 5096 FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS substrates with an unbranched Glc residue at the )1 site and catalyzes cleavage of the glycosidic bond of unbranched Glc residues. Activity of the XEG mutant Y457G To confirm the role of Tyr457, we constructed a XEG variant, Y457G, which was expressed in Escherichia coli, purified, and characterized. The kinetic constants of Y457G against tamarind seed xyloglucan were determined. The apparent K m values of the wild-type and Y457G enzymes were 0.47 and 0.43 mgÆmL )1 , respectively, and their specific activities were 15.7 and 3.97 unitÆmg )1 protein, respectively. Next, the substrate specificity was investigated using a tetradecasaccharide, XXXGXXXG. Wild-type XEG generated only XXXG (Fig. 4), because of its strict specificity for glycosidic bonds of the unbranched Glc residue. By contrast, the Fig. 2. Schematic drawing of the entire structures (traces of Ca atoms) of XEG (A), the OXG-RCBH ⁄ substrate (XXXG) complex (B) and the Xgh74A ⁄ substrate (XLLG and XXLG) complex (C). The substrates are shown as stick models. The exo loop of OXG-RCBH (Gly375–His385) is shown in red. K. Yaoi et al. Key amino acid residue of Geotrichum XEG FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS 5097 Y457G mutant released various oligosaccharides from cleavage of XXXGXXXG. The structures of these products were X, XG, GX, XX, XXG, XXX and XXXG, as confirmed by MALDI-TOF MS combined with enzymatic treatments of the reaction products using isoprimeverose-producing oligoxyloglucan hydro- lase from Oerskovia [14] and b-glucosidase from almond, Prunus dulcis, as described previously [8] (data not shown). On the basis of the product patterns, we propose the possible cleavage sites shown in Fig. 4. Therefore, the Y457G variant can catalyze cleavage of the glycosidic bond of branched Glc residues, i.e. the Glc residue at the )1 site can be branched. The Y457G mutant enzyme was less selective, indicating that Y457 plays an important role in determining sub- strate specificity. Conclusion Geotrichum M128 produces two GH74 enzymes, XEG and OXG-RCBH. They share 48% amino acid identity and three-dimensional structures. The majority of the residues interacting with the substrate are well-con- served. However, the residues that determine substrate specificity and mode of action were distinct. XEG and OXG-RCBH display endo and exo modes of action, respectively. A loop structure that determines the exo action of OXG-RCBH is not present in XEG. Fig. 3. Comparison of the active site from )2 to +2 for XEG and the OXG-RCBH ⁄ XXXG complex. The stereo views of XEG and OXG-RCBH are shown in blue and purple, respectively. The substrate XXXG and the amino acids that interact with the substrate are shown as stick models. The substrate is colored blue, light green, green and orange at the )2, )1, +1 and +2 sites, respectively. The exo loop of OXG- RCBH is colored in red. Fig. 4. HPLC analysis of the digestion prod- ucts of the xyloglucan oligosaccharide XXXGXXXG by wild-type XEG and the Y457G mutant. The xyloglucan oligosaccha- ride (XXXGXXXG) was incubated with wild- type XEG or the Y457G mutant, and the products were analyzed by HPLC. The pro- posed cleavage sites are indicated by arrows. (A) Oligosaccharide marker. (B) Products from cleavage by wild-type XEG. (C) Products from cleavage by the Y457G mutant. Key amino acid residue of Geotrichum XEG K. Yaoi et al. 5098 FEBS Journal 276 (2009) 5094–5100 ª 2009 The Authors Journal compilation ª 2009 FEBS A further difference in the active site residues involves recognition of a Xyl side chain at the )1 site. OXG-RCBH recognizes a Xyl side chain at the )1 site (Asn488), whereas XEG does not. In XEG, residue Tyr457, which corresponds to Gly464 of OXG-RCBH, appears to protrude into the )1 site, providing a basis for the substrate specificity of XEG. In fact, a single amino acid substitution of Tyr457 by Gly caused a significant change in substrate recogni- tion by XEG. These results suggest that the substrate specificities of XEG and OXG-RCBH depend on a limited number of residues in the substrate binding cleft. Experimental procedures Purification of XEG The gene encoding the mature region of XEG [7] was subcl- oned into pET14-b (Novagen, Madison, WI, USA) using NdeI and BamHI sites, resulting in fusion of a His 6 tag to the N-terminus. The resultant product was transformed into E. coli BL21-CodonPlus (DE3) RP (Stratagene, La Jolla, CA, USA). Expression was induced by addition of isopropyl-b-d-thio-galactopyranoside (final concentration 0.1 mm) for 16 h at 20 °C. The soluble, intracellular recom- binant protein was extracted using BugBuster protein extraction reagent (Novagen), and was purified using a HiTrapÔ chelating column (GE Healthcare, Little Chal- font, UK). Solid ammonium sulfate was added to the active fractions from the column to a final concentration of 0.75 m, and the fractions were loaded onto a Resource PHE hydrophobic interaction chromatography column (Amersham Biosciences) equilibrated with 25 mm imidaz- ole ⁄ HCl buffer (pH 7.4) containing 0.75 m (NH 4 ) 2 SO 4 . Bound proteins were eluted using a linear gradient of 0.75–0 m (NH 4 ) 2 SO 4 in 25 mm imidazole ⁄ HCl buffer (pH 7.4). Finally, XEG was resolved on a HiLoad 16 ⁄ 60 Superdex 200 pg column (Amersham Biosciences) in 25 mm imidazole ⁄ HCl buffer (pH 7.4). Before crystallization, purified XEG was concentrated to 8 mgÆmL )1 using an Ultrafree-15 centrifugal filter device (Millipore, Bedford, MA, USA). Crystal structure determination of XEG Crystallization of XEG was performed at 20 °C using the hanging-drop vapor-diffusion method [15]. The crystalliza- tion conditions were initially screened using the screens Index and PEG ⁄ Ion (both Hampton Research, Aliso Viejo, CA), and Wizard I, Cryo I and Cryo II (all DeCODE Genetics, Reykjavik, Iceland), and were refined by varying the pH of the buffer and the concentration of the precipi- tant. Prior to data collection, a crystal was transferred into Paratone-N (Hampton Research) and mounted on a CryoLoop (Hampton Research) of 20 lm diameter. The mounted crystal was immersed in liquid nitrogen. Diffrac- tion data were collected at 100 K on Beamline BL44B2 at SPring-8 (Harima Institute, Hyogo, Japan) using an ADSC Quantum 210 CCD detector (Area Detector Systems Corporation, Poway, CA, USA) with 1.0000 A ˚ radiation, and were processed using HKL2000 [16] and the ccp 4 pro- gram suite [17]. The structure of XEG was solved by means of the molecular replacement method, using the AMORE program [18] in the ccp 4 package. The coor- dinates of OXG-RCBH (PDB code 1sqj) were used as a search model. The cns program [19] was used for refinement against the 20–2.5 A ˚ intensity data. A ran- domly selected portion of the diffraction data (5.0%) was used to calculate the free R factor [20]. The pro- gram coot [21] was used to display and correct the structure. Figures were created using pymol (DeLano Scientific LLC, Palo Alto, CA; http://www. pymol.org). The coordinates were deposited in the Pro- tein Data Bank (3A0F). Construction of the Y457G mutant The Y457G mutant was constructed using the QuikChangeÒ procedure (Stratagene) with primers 5¢-TCTTCAGCGGC ATGGGCGACCTCGGCGGCAT-3¢ and 5¢-ATGCCGC CGAGGTCGCCCATGCCGCTGAAGA-3¢. The recombi- nant protein was purified using a HiTrapÔ chelating column and Resource PHE column (both Amersham Biosciences). Analysis of kinetic parameters The kinetic parameters were determined at various concen- trations of the substrate, tamarind seed xyloglucan (Dainip- pon Sumitomo Pharma, Osaka, Japan), in 50 mm sodium acetate buffer (pH 5.5) at 45 °C for 15 min. The bicinchoni- nate assay was used to quantify reducing sugars. The Michaelis constant (K m ) and specific activity were calcu- lated from a plot of initial reaction rates versus substrate concentration using Prism (GraphPad Software, San Diego, CA, USA). One unit was defined as the amount of enzyme that released 1 lmol of glucose equivalent as reducing sug- ars from xyloglucan per minute. Analysis of substrate specificity Substrate specificity was analyzed using xyloglucan tetra- decasaccharide, XXXGXXXG, prepared as described previ- ously [8]. The oligosaccharide (0.2 mg) was incubated with each enzyme in 20 lLof50mm sodium acetate buffer (pH 5.5) at 45 °C for 16 h. 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The crystal structure of a xyloglucan-specific endo- b -1,4- glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate. precise anal- ysis of the mode of action of Xgh7 4A has not been performed, Xgh7 4A appears to be an endoglucanase because it has an open cleft. Comparison of the