Nội dung

An investigation of the substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina Tom Desmet1, Tineke Cantaert1, Peter Gualfetti2, Wim Nerinckx1, Laurie Gross2, Colin Mitchinson2 and Kathleen Piens1 1 Department of Biochemistry, Physiology and Microbiology, Faculty of Sciences, Ghent University, Belgium 2 Genencor International Inc., Palo Alto, CA, USA Keywords Cel74A; glycoside hydrolase; Hypocrea jecorina; Trichoderma reesei; xyloglucanase Correspondence T. Desmet, Department of Biochemistry, Physiology and Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium Fax: +32 9 264 5332 Tel: +32 9 264 5272 E-mail: t.desmet@ugent.be (Received 2 August 2006, revised 21 October 2006, accepted 9 November 2006) The substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina (Trichoderma reesei) was examined using several polysaccharides and oligosaccharides. Our results revealed that xyloglucan chains are hydrolyzed at substituted Glc residues, in contrast to the action of all known xyloglucan endoglucanases (EC 3.2.1.151). The building block of xyloglucan, XXXG (where X is a substituted Glc residue, and G is an unsubstituted Glc residue), was rapidly degraded to XX and XG (kcat ¼ 7.2 s)1 and Km ¼ 120 lm at 37 C and pH 5), which has only been observed before with the oligoxyloglucan-reducing-end-specific cellobiohydrolase from Geotrichum (EC 3.2.1.150). However, the cellobiohydrolase can only release XG from XXXGXXXG, whereas Cel74A hydrolyzed this substrate at both chain ends, resulting in XGXX. Differences in the length of a specific loop at subsite + 2 are discussed as being the basis for the divergent specificity of these xyloglucanases. doi:10.1111/j.1742-4658.2006.05582.x The tropical soft rot fungus Hypocrea jecorina (formerly Trichoderma reesei) secretes one of the most efficient and best characterized mixtures of cellulolytic enzymes, including at least five endoglucanases (EG; EC 3.2.1.4) and two exoglucanases or cellobiohydrolases (CBH, EC 3.2.1.91). These enzymes are divided into different glycoside hydrolase (GH) families on the basis of sequence similarities and consequent conservation of fold, and stereochemical outcome of the reaction: inversion (single displacement) or retention (double displacement) of the anomeric configuration [1,2]. A regularly updated overview of the different GH families can be found at http://afmb.cnrs-mrs.fr/ CAZY/index.html [3]. Several new genes coding for biomass-degrading enzymes have been identified in H. jecorina [4]. One of these enzymes is Cel74A, formerly EG VI [5], which is coregulated with known cellulases [4] and has recently been characterized as a xyloglucanase [6]. Specific xyloglucanases represent a relatively new class of enzymes for which important issues, such as nomenclature and EC numbering, still have to be addressed [6]. Little is known about the structural factors that contribute to xyloglucanase activity, as only one enzyme–xyloglucan complex has so far been studied by crystallography [7]. Although EGs from various GH families have been shown to display substantial activity on xyloglucan, specific xyloglucanases can be found in GH families 5, 12, 16, 26 and 74. The last of these currently comprises 24 sequences, including Cel74A, but only eight members have been characterized [6–13]. An inverting mechanism has been demonstrated for GH family 74 [11], and the three-dimensional structures of two representative enzymes have been published [7,14]. Xyloglucan is a hemicellulose composed of a b-1,4d-GlcP backbone with regularly distributed a-d-XylP Abbreviations CNP, 2-chloro-4-nitrophenol; DSC, differential scanning calorimetry; EG, endoglucanase; EndoH, endoglycosidase H; GH, glycoside hydrolase; HPAEC-PAD, high-pressure anion exchange chromatography with pulsed amperometric detection; OXG-RCBH, oligoxyloglucan reducing-endspecific cellobiohydrolase; Xyl, xylose. 356 FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS T. Desmet et al. (Xyl ¼ xylose) substitutions at C6. A specific letter code has been proposed, in which the unsubstituted and substituted Glc residues are represented as G and X, respectively [15]. Further substitution with b-d-GalP at C2 of a Xyl residue results in a trisaccharide represented as the letter L. The repeating unit in xyloglucan from Tamarindus indica has been identified as XXXG, with Gal substitutions occurring only at the middle two positions [16]. Cel74A from H. jecorina has been reported to hydrolyze this polymer at unsubstituted Glc residues, resulting in the release of XXXG units [6]. However, the hydrolysis of this building block by the GH family 74 oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) from Geotrichum sp. M128 [8] has prompted us to examine the specificity of Cel74A using xyloglucan oligosaccharides. In this article, hydrolysis at substituted Glc residues is clearly demonstrated, and a new interpretation of the endproducts of xyloglucan hydrolysis is presented. A subsite map of Cel74A is proposed, and the structure– function implications for GH family 74 xyloglucanases are discussed. Results and Discussion Protein characterization Cel74A was purified to apparent homogeneity from an engineered strain of H. jecorina by a combination of gel filtration and affinity chromatography. The single band observed with SDS ⁄ PAGE corresponds to a molecular mass of about 100 kDa (Fig. 1), in contrast to the theoretical value of 85.070 kDa for the mature protein (SwissProt Q7Z9M8), consisting of a catalytic domain, a linker region, and a C-terminal carbohydrate-binding module. A high apparent molecular mass for intact Cel74A (105 kDa) was also reported by Grishutin et al. [6]. Their enzyme sample, however, also contained lower molecular mass species (75– 90 kDa), whereas heterogeneity was not observed in our Cel74A preparation. It has long been known that glycosylated proteins can run with aberrantly low mobility on SDS ⁄ PAGE [17]. There are two potential N-glycosylation sites in Cel74A (N213 and N417), but the purified protein was not extensively N-glycosylated, as treatment with endoglycosidase H (EndoH) did not result in a reduction of the apparent molecular mass (Fig. 1). Chymotryptic peptide mapping and MS ⁄ MS not only identified the protein as full-length Cel74A, giving over 80% coverage, including the expected N-terminus and C-terminus, but also revealed that both N-glycosylation sites carry a single GlcNAc after EndoH treatment (data Hypocrea jecorina Cel74A 1 2 3 4 200 116 97 66 45 Fig. 1. SDS ⁄ PAGE analysis of Cel74A: lane 1, molecular mass markers (kDa); lane 2, Cel74A; lane 3, Cel74A treated with EndoH; lane 4, EndoH. not shown). MS of the EndoH-treated Cel74A yielded a molecular mass of 86.552 kDa. Taking into account the two GlcNAc residues, this observed molecular mass exceeds the calculated molecular mass by 1.075 kDa, a mass consistent with six hexose residues. GHs containing a carbohydrate-binding module are routinely O-glycosylated in the linker region between the catalytic domain and the carbohydrate-binding module. Regions 725–739 and 764–782 of the Cel74A linker were not identified in the chymotryptic map and, presumably, contain the sites of O-glycosylation, but we have not identified the specific site(s) or the form of the O-glycans. The CD spectrum of Cel74A looks a lot like that of an unordered protein (Fig. 2) [18]. Addition of substrate does not affect the overall secondary structure but decreases the ellipticity around 230 nm that is diagnostic of tryptophan exciton coupling [19]. The structure of GH family 74 enzymes has recently been shown to consist of two b-propeller domains [14]. Such a fold has been recognized in a wide range of proteins, including other GHs (clans E, F and J) [3] and hemopexin [20]. The CD spectrum of the latter protein [21] is very similar to the one observed here; therefore, the unusual spectrum is probably diagnostic of the b-propeller fold. Thermal melting can be monitored at 212 nm and reveals a Tm of 64.1 C at pH 5.5, coincident with the value of 64.0 C determined by differential scanning calorimetry (DSC) (data not shown). The pH of optimum stability as determined by DSC is 5 (data not shown). FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 357 Hypocrea jecorina Cel74A T. Desmet et al. triose (5% decrease in activity) (for conditions, see Experimental procedures). The accommodation of a b-1,3-bond between subsites ) 1 and + 1 is unusual for a b-1,4-glucanase but not unprecedented [22], and is supported by the inhibition of Cel74A by laminaritriose (10% decrease in activity on XXXG). Mixed-linkage oligosaccharides, however, seem to be preferentially cleaved at b-1,4-bonds (Table 2). Hydrolysis of xyloglucan chains Fig. 2. CD spectra of Cel74A at 25 C and pH 5.0, with (dashed line) and without (solid line) the addition of 0.1 mgÆmL)1 tamarind xyloglucan. Data were collected every 1 nm for 5 s, and three spectra were averaged. Substrate specificity The hydrolytic activity of Cel74A on cellotetraose was optimal at pH 5 and 60 C (data not shown). The kinetic parameters for the hydrolysis of carboxymethyl cellulose, b-glucan and xyloglucan (Table 1) differ from those reported by Grishutin et al. [6], although the strong preference of Cel74A for the last of these substrates is clear in both studies. A possible reason for the quantitative differences is the presence of truncated protein in the previously reported enzyme sample. The considerable increase in activity on cellotetraose and 2-chloro-4-nitrophenyl cellotrioside (GGGCNP) (CNP, 2-chloro-4-nitrophenol) in comparison to cellotriose and GGCNP, respectively (Table 2), suggests an active center composed of at least four subsites () 2 ⁄ + 2). The preferential release of GCNP from the chromogenic substrates excludes the use of direct spectrophotometric assays. The strong contribution of all four subsites to ligand binding is reflected in the higher degree of inhibition of activity on XXXG by cellotetraose (60% decrease in activity) compared to cello- The end-products of xyloglucan hydrolysis by Cel74A have been reported to be XXXG, XXLG ⁄ XLXG and XLLG (see introduction for nomenclature), indicating hydrolysis at nonsubstituted Glc residues [6]. However, we demonstrate by high-pressure anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Fig. 3A) and ESI-MS (not shown) that XXXG is readily hydrolyzed to XX and XG by Cel74A. Moreover, this enzyme exhibits 30-fold higher activity on XXXG (kcat ¼ 7.2 s)1 and Km ¼ 120 lm) than on GGGG (Table 2). Its active center must therefore contain specific binding sites for the Xyl residues at subsites ) 2 to + 1 (Fig. 4), as has been proposed for the GH family 74 exoglucanase from Geotrichum [8]. With such an active site composition, one would expect the end-products of xyloglucan hydrolysis by Cel74A to be XGXX, XGXL ⁄ LGXX and LGXL instead of their structural isomers XXXG, XXLG ⁄ XLXG and XLLG, respectively. Unfortunately, these isomers cannot be discriminated by MS and are undoubtedly very hard to separate by HPLC. They would be indistinguishable on the chromatograms reported by Grishutin et al., as the two octasaccharide isomers present in their product mixture also coeluted [6]. Further proof of hydrolysis at substituted Glc residues by Cel74A was obtained with the oligosaccharide XXXGXXXG (Figs 3 and 5). Although this compound has been treated with b-galactosidase by the supplier, some b-1,2-GalP residues are still present, Table 1. Kinetic parameters for the hydrolysis of b-glucans by Cel74A (37 C, pH 5). ND, not determined (very low activity); PASC, phosphoric acid-swollen cellulose; CMC, carboxymethyl cellulose; HEC, hydroxyethyl cellulose. Substrate Backbone kcat (s)1) Km (mgÆmL)1) kcat ⁄ Km (mLÆmg)1Æs)1) Avicel PASC CMC HEC Laminarin b-Glucan Xyloglucan b-1,4b-1,4b-1,4b-1,4b-1,3b-1,3 ⁄ 1,4b-1,4- ND 7 52 138 193 306 805 ND 0.8 ± 1.3 ± 4.8 ± 2.1 ± 0.5 ± 0.3 ± ND 9 40 29 92 612 2683 358 ± ± ± ± ± ± 2 8 11 23 9 13 0.2 0.3 1.2 0.6 0.1 0.1 FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS T. Desmet et al. Hypocrea jecorina Cel74A Table 2. Relative activity of Cel74A towards oligosaccharides (37 C, pH 5). Substratea Relative activity GGCNP GGGCNP GGGGCNP GGG GGGG GGGGG G3G3G GG3G GGG3G XXXG XXXGXXXG 0.24 0.68 0.84 0.32 0.76 1 0.38 0.52 0.83 27 39 Final products G + GCNP GG + GCNP GGG + GCNP G + GG GG + GG GGG + GG G + G3G G + G3G GG + G3G XX + XG XX + XGXX + XG Fig. 4. Schematic representation of the active site () 2 ⁄ + 2) of Cel74A from H. jecorina. a X, Glc substituted with a-D-XylP at C6; CNP, 2-chloro-4-nitrophenol. The number 3 indicates a b-1,3 bond; all other backbone linkages are b-1,4. Fig. 3. HPAEC-PAD profiles of xyloglucan oligosaccharides: XXXG (pure) after incubation with Cel74A (A), and XXXGXXXG (impure) before (B) and after incubation with Cel74A (C) or with Cel12A (D). The numbering of the peaks is as follows: 1, XG; 2, XX; 3, XXXG; 4, XGXX; 5, XXXGXX ⁄ XGXXXG; 6, XXXGXXXG. Some of the compounds in (B)–(D) appear as a cluster of peaks, due to partial substitution with Gal residues. and therefore the substrate and several products appear as a cluster of peaks on the chromatograms (Fig. 3B–D). Attempts to treat the sample more extensively with b-galactosidase from Aspergillus niger resulted in general degradation, presumably by contaminating enzyme activities. Nevertheless, this substrate mixture is still very useful for the qualitative analysis of the degradation pattern, and will be referred to as XXXGXXXG for simplicity. From the results presented here, it is obvious that XXXGXXXG is rapidly hydrolyzed to XX, XGXX and XG. Indeed, the release of the small products (XG ⁄ XX) allows the heptasaccharide product to be identified as XGXX (Fig. 3C). The unambiguous detection by ESI-MS of XGXXXG as intermediate product (Fig. 5) confirms that the substrate is not hydrolyzed to its repeating unit XXXG. Moreover, it can be concluded that Cel74A is not able to hydrolyze xyloglucan at unbranched Glc residues, as XGXX does not disappear after hours of incubation with Cel74A (data not shown), in agreement with the size of the end-products of xyloglucan hydrolysis reported by Grishutin et al. [6]. As a comparison, XXXGXXXG was treated with H. jecorina Cel12A (EG III), which is known to hydrolyze xyloglucan at unbranched Glc residues, thus releasing XXXG units [23]. Indeed, this cellulase is not able to hydrolyze XXXG (data not shown) and only produces a heptasaccharide product from XXXGXXXG (Fig. 3D). Cel12A and Cel74A clearly display different degradation patterns on xyloglucan chains. Mode of action The recent elucidation by X-ray crystallography of the structure of two GH family 74 enzymes has suggested a structural basis for the difference between an exoglucanase (OXG-RCBH from Geotrichum) [14] and an EG (XGH74A from Clostridium thermocellum) [7]. Both enzymes have an active site located in an open cleft, but a specific loop segment that blocks the entrance of the cleft in OXG-RCBH at subsite + 2 could provide this enzyme with exoglucanase activity by preventing binding to the middle of a xyloglucan FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 359 Hypocrea jecorina Cel74A T. Desmet et al. 1076.2 100 % A C B D 554.0 1157.8 0 629.1 100 554.0 497.0 929.2 % 1076.2 782.1 0 500 1000 m/z Fig. 5. ESI-MS analysis of XXXGXXXG, before (A) and after (B) incubation with Cel74A, and the proposed cleavage pattern (C) with the theoretical m ⁄ z value (z in superscript) of the most important fragments (the position of the Gal residues is arbitrary) detected as Na+ adducts (D). chain. The ‘exo-loop’ sequence is absent in XGH74A and all other characterized GH family 74 EGs, but part of it (seven out of 11 amino acids) is conserved in H. jecorina Cel74A (SwissProt Q7Z9M8). OXG-RCBH hydrolyzes XXXG to XX and XG, just like Cel74A, but it cleaves XXXGXXXG exclusively at the reducing end [8], in contrast to the presently studied enzyme (Table 2). Cel74A has been shown to lower the average molecular mass of xyloglucan very slowly, which is typical for an exo mode of action [6]. However, this exo-like behavior is probably not absolute [6] nor reducing-end-specific, in light of the fast hydrolysis of XXXGXXXG at both chain ends (Figs 3 and 5). Indeed, the exo-loop cannot completely block the active site cleft of Cel74A at subsite + 2, as its end-products of xyloglucan hydrolysis have a backbone degree of polymerization of four instead of two. Furthermore, its preference for soluble (carboxymethylcellulose ⁄ hydroxyethyl cellulose) over crystalline (Avicel) and amorphous (phosphoric acidswollen cellulose) is typical of EG activity (Table 1). A possible conformation of XXXG in subsites ) 2 ⁄ + 2 of OXG-RCBH is shown in Fig. 6. The exoloop is located roughly above the postulated + 2 subsite, where it is believed to restrict binding of a branched Glc residue [14]. Docking experiments on OXG-RCBH indicate that the shorter loop length in Cel74A can be expected to increase access of an oligosaccharide to potential subsites + 3 ⁄ + 4, and may be the basis for the unique mode of action of this enzyme. More definitive statements await the solution of the Cel74A structure, which is currently underway. 360 Fig. 6. Possible conformation of XXXG in subsites ) 2 ⁄ + 2 of OXGRCBH from Geotrichum (Protein Data Bank 1sqj). The glucosyl unit in subsite ) 1 was minimized in a skew-boat 1S3-conformation, by analogy with the substrate distortion often observed in crystallographic studies of b-glucanase complexes [2]. Conclusions The xyloglucanase Cel74A from H. jecorina is shown in this study to release XGXX units from xyloglucan chains, in contrast to a recently published report [6]. Assuming hydrolysis at unsubstituted Glc residues, those authors have tentatively identified the heptasaccharide product of xyloglucan hydrolysis as XXXG. Indeed, analysis by HPLC and ESI-MS does not discriminate between these heptasaccharide isomers. However, the fast hydrolysis of XXXG by Cel74A (kcat ¼ 7.2 s)1 and Km ¼ 120 lm) and the release of both XX and XG from XXXGXXXG (Figs 3 and 5) FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS T. Desmet et al. revealed in our study clearly indicate cleavage at substituted Glc residues. An active site composed of at least four subsites () 2 ⁄ + 2) and containing additional interaction sites for the Xyl residues is proposed for Cel74A (Fig. 4), in accordance with OXG-RCBH from Geotrichum [8]. The latter enzyme hydrolyzes xyloglucan oligosaccharides at substituted Glc residues, but releases only XG from XXXGXXXG and cannot be very active on xyloglucan, in contrast to Cel74A (Table 1). A specific loop at subsite + 2 is believed to be responsible for the exoglucanase-like behavior of Cel74A and OXG-RCBH, but the exact mechanism is not yet clear. This ‘exo-loop’ also seems to restrict the access of branched residues to subsite + 2, and could therefore be the major determinant of the degradation pattern of GH family 74 xyloglucanases. Enzymes that hydrolyze xyloglucan at branched Glc residues obviously need a ) 1 subsite that is relatively spacious (Fig. 6), but a spacious ) 1 subsite is also observed in the EG XGH74A from Clostridium thermocellum, which hydrolyzes xyloglucan at unbranched Glc residues [7]. Clearly, further studies are required to determine the exact nature of the interactions of the loop residues in different GH family 74 enzymes, especially with branched substrates. Access to more crystal structures of enzyme–ligand complexes will hopefully lead to a better understanding of the factors that contribute to the divergent specificity of these xyloglucanases. Experimental procedures Enzyme expression and purification Cel74A was obtained from an engineered derivative of H. jecorina strain RL-P37, in which four cellulase genes (cbh1 ⁄ cel7a, cbh2 ⁄ cel6a, egl1 ⁄ cel7b, and egl2 ⁄ cel5a) were disrupted as described in Bower et al. [24]. Higher levels of Cel74A expression were observed in a derivative of this strain, transformed with a circular plasmid carrying the catalytic domain of Cel7A (carbohydrate-binding module I) behind the cel7a promoter. The resultant strain was grown at 25 C in a batch-fed process with lactose as carbon source and inducer, using a minimal fermentation medium essentially as described in Ilmen et al. [25]. First, 0.8 L of medium containing 5% glucose was inoculated with 1.5 mL of spore suspension. After 48 h, the culture was transferred to 6.2 L of the same medium in a 14 L fermenter (Biolafitte, Princeton, NJ, USA). One hour after the glucose was exhausted, a 25% (w ⁄ w) lactose feed was started in a carbon-limiting fashion, so as to prevent its accumulation. The pH during fermentation was maintained in the range Hypocrea jecorina Cel74A 4.5–5.5. The final protein concentration in the culture supernatant was 12.4 gÆL)1. After concentration of the supernatant to 88 gÆL)1 by ultrafiltration at 4 C with a PTGC membrane from Millipore (Billerica, MA, USA), Cel74A was purified by gel filtration followed by affinity chromatography. A sample of 6.5 mL was applied to a 2.6 · 60 cm Superdex 75 preparative grade column (Amersham Biosciences, Piscataway, NJ, USA) equilibrated in 0.15 m sodium acetate ⁄ acetic acid (pH 5.5). Cel74A eluted in the void volume, with 95% purity as estimated by SDS ⁄ PAGE (not shown). Subsequent affinity chromatography, as described in Tomme et al. [26], succesfully removed residual b-glucosidase activity and yielded an electrophoretically homogeneous preparation. The final sample was concentrated with an Ultrafree-MC Centrifugal Filter Unit (Millipore) to an A280 of 5, and was stored at 4 C in MilliQ-water (Ultrapure Water System; Millipore). Protein characterization The concentration of Cel74A was determined from the absorbance at 280 nm, using an extinction coefficient of 200 900 m)1Æcm)1 calculated on the basis of the amino acid composition [27]. EndoH (Sigma-Aldrich, St Louis, MO, USA) treatment of Cel74A was performed according to the supplied protocol. ESI-MS was performed with the LCQ Classic and Advantage (Thermo Electron Corporation, Waltham, MA, USA) for the determination of the molecular mass and for peptide mapping, respectively. CD spectra were collected from a 0.1 cm path length cell on an Aviv 215 spectrophotometer (Proterion Corporation, Piscataway, NJ, USA) equipped with a five-position thermoelectric cell holder. The CD thermal denaturation experiments were performed at 212 nm, where the maximum signal difference occurs. The data were fitted to a two-state transition [28] using savuka software provided by O Bilsel (University of Massachusetts Medical School, N. Worcester, MA, USA). The midpoint of the transition (Tm) is an apparent value, as the thermal denaturation was not reversible and was accompanied by precipitation. DSC thermograms were collected on a VP DSC instrument from Microcal (Northampton, MA, USA). The thermal denaturation of Cel74A was completely irreversible, and no transition was seen in a repeat scan; therefore, an apparent Tm was approximated as the midpoint of the DSC peak. Substrate specificity b-Galactosidase from A. niger, tamarind xyloglucan and the derived oligosaccharide XXXG were obtained from Megazyme (Bray, Co. Wicklow, Ireland), barley b-glucan, laminarin, laminaritriose, carboxymethyl cellulose, hydroxy- FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 361 Hypocrea jecorina Cel74A T. Desmet et al. ethylcellulose and Avicel were obtained from SigmaAldrich, and the cello-oligosaccharides were obtained from Merck (Darmstadt, Germany). The oligosaccharides derived from b-glucan were a gift from A Planas (Universitat Raman Llull, Barcelona, Spain), and the xyloglucan oligosaccharide XXXGXXXG was a gift from B McCleary (Megazyme). Phosphoric acid-swollen cellulose was prepared according to Wood [29], and the CNP glycosides were synthesized as in Van Tilbeurgh [30]. All reactions were performed at 37 C in 0.1 m sodium acetate ⁄ acetic acid buffer (pH 5), except for the determination of the pH profile (McIlvaine buffers pH 3.5–7) and the temperature profile (30–80 C) for cellotetraose hydrolysis. The increase in reducing sugars was measured by the bicinchoninic acid method described by Mopper and Gindler [31], using a Microplate Reader 550 from Bio-Rad (Hercules, CA, USA). A standard curve was obtained using 0–100 lm glucose. The kinetic parameters for the hydrolysis of the different polymers were determined by mixing 0.01–10 mgÆmL)1 substrate with 14–140 nm enzyme. Release of soluble reducing sugars was measured by the bicinchoninic method as above. The relative activity on the different oligosaccharides and chromogenic glycosides was determined using 2 mm substrate and 2 lm enzyme. At regular intervals, samples were deactivated by boiling for 2 min. After 10-fold dilution in water, the oligosaccharides were analyzed by HPAEC-PAD (Dionex, Sunnyvale, CA, USA), and the chromogenic glycosides by normal-phase HPLC (Waters, Milford, MA, USA). ESI-MS of the reaction products was performed with a Q-trap mass spectrometer in positive mode (Applied Biosystems, Foster City, CA, USA). The kinetic parameters for the hydrolysis of 0.04–4 mm XXXG were determined by HPAEC-PAD with 0.1 lm enzyme. A calibration curve was obtained using 0–100 lm substrate. The degree of inhibition by oligosaccharides was determined by comparing the activity of Cel74A on 0.1 mm XXXG, determined as described above, in the presence and absence of 2 mm inhibitor. The inhibitors were not hydrolyzed in the time course of these experiments (10 min). Docking experiments Docking of the oligosaccharide XXXG into the active site of OXG-RCBH from Geotrichum (Protein Data Bank 1sqj) was carried out with autodock version 3.0.5 [32]. The ligand was drawn and minimized with hyperchem 4.5 (MM + force field; HyperCube Inc, Gainesville, USA) [33,34]. The glucose unit in subsite ) 1 was drawn in a skew-boat 1S3 conformation, by analogy with the substrate distortion often found in crystallographic studies of b-glucanase complexes [2]. The graphical user interface autodocktools (ADT 1.1) was used for formatting of the ligand and macromolecule, as well as for setting the grid and docking parameters. Minor variations of the standard 362 Lamarckian genetic algorithm parameter settings were used: the number of runs was set at 50, with a run termination of 7000 generations at a maximum of 25 · 107 energy evaluations. The colored figure was prepared with pymol-osx 0.93 (http://www.pymol.org). Acknowledgements The authors wish to thank Chris Cummings (Genencor) for H. jecorina strain construction, Nicole Chow (Genencor) for peptide mapping, Koen Sandra (Ghent University) for ESI-MS analyses of reaction products, and Marc Claeyssens (Ghent University) for fruitful discussions. This work was supported in part by a subcontract from The Office of Biomass Program, within the DOE Office of Energy Efficiency and Renewable Energy. Tom Desmet holds a fellowship of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen). References 1 Davies G & Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3, 853–859. 2 Vasella A, Davies GJ & Bohm M (2002) Glycosidase mechanisms. Curr Opin Chem Biol 6, 619–629. 3 Coutinho PM & Henrissat B (1999) Carbohydrateactive enzymes: an integrated database approach. In Recent Advances in Carbohydrate Bioengineering (Gilbert HJ, Davies G, Henrissat B & Svensson B, eds), pp. 3–12. Royal Society of Chemistry, Cambridge. 4 Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS et al. (2003) Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278, 31988–31997. 5 Bower BS, Clarkson KA, Collier KD, Kellis JT, Kelly MB & Larenas EA (2003) High molecular weight Trichoderma cellulase. EP 0934402, B1 Patent. 6 Grishutin SG, Gusakov AV, Markov AV, Ustinov BB, Semenova M & Sinitsyn AP (2004) Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochim Biophys Acta 1674, 268–281. 7 Martinez-Fleites C, Guerreiro CIPD, Baumann MJ, Taylor EJ, Prates JAM, Ferreira LMA, Fontes CMGA, Brumer H & Davies GJ (2006) Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation. J Biol Chem 281, 24922–24933. 8 Yaoi K & Mitsuishi Y (2002) Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase. J Biol Chem 277, 48276–48281. FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS T. Desmet et al. 9 Hasper AA, Dekkers E, van Mil M, van de Vondervoort PJI & de Graaff LH (2002) EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan. Appl Environ Microbiol 68, 1556– 1560. 10 Chhabra SR & Kelly RM (2002) Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM). FEBS Lett 531, 375–380. 11 Irwin DC, Cheng M, Xiang B, Rose JKC & Wilson DB (2003) Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca. Eur J Biochem 270, 3083–3091. 12 Yaoi K & Mitsuishi Y (2004) Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128. FEBS Lett 560, 45–50. 13 Yaoi K, Nakai T, Kameda Y, Hiyoshi A & Mitsuishi Y (2005) Cloning and characterisation of two xyloglucanases from Paenobacillus sp. strain KM21. Appl Environ Microbiol 71, 7670–7678. 14 Yaoi K, Kondo H, Noro N, Suzuki M, Tsuda S & Mitsuishi Y (2004) Tandem repeat of a seven-bladed b-propeller domain in oligoxyloglucan reducing-end-specific cellobiohydrolase. Structure 12, 1209–1217. 15 Fry SC, York WS, Albersheim P, Darvill A, Hayashi T, Joseleau JP, Kato Y, Lorences EP, Maclachlan GA, McNeil M et al. (1993) An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol Plant 89, 1–3. 16 Vincken J-P, York WS, Beldman G & Voragen AGJ (1997) Two general branching patterns of xyloglucan, XXXG and XXGG. Plant Physiol 114, 9–13. 17 Segrest JP & Jackson RL (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Methods Enzymol 28, 54–63. 18 Venyaminov SY & Yang JT (1996) Determination of protein secondary structure. In Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman GD, ed.), pp. 69–107. Plenum Press, New York. 19 Woody RW & Dunker AK (1996) Aromatic and cystine side-chain circular dichroism in proteins. In Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman GD, ed.), pp. 109–157. Plenum Press, New York, NY. 20 Paoli M, Anderson BF, Baker HM, Morgan WT, Smith A & Baker EN (1999) Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-propeller domains. Nat Struct Biol 6, 926–931. 21 Wu ML & Morgan WT (1993) Characterization of hemopexin and its interaction with heme by differential Hypocrea jecorina Cel74A 22 23 24 25 26 27 28 29 30 31 32 33 34 scanning calorimetry and circular-dichroism. Biochemistry 32, 7216–7222. Sandgren M, Berglund GI, Shaw A, Ståhlberg J, Kenne L, Desmet T & Mitchinson C (2004) Crystal complex structures reveal how substrate is bound in the ) 4 to the + 2 binding sites of Humicola grisea Cel12A. J Mol Biol 342, 1505–1517. Vincken JP, Beldman G & Voragen AG (1997) Substrate specificity of endoglucanases: what determines xyloglucanase activity? Carbohydr Res 298, 299–310. Bower B, Kodama K, Swanson B, Fowler T, Meerman H, Collier K, Mitchinson C & Ward M (1998) Hyperexpression and glycosylation of Trichoderma reesei EGIII. In Carbohydrases from Trichoderma reesei and Other Micro-organisms (Claeyssens M, Nerinckx W & Piens K, eds), pp. 327–334. Royal Society of Chemistry, Cambridge. Ilmen M, Saloheimo A, Onnela ML & Penttila ME (1997) Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63, 1298–1306. Tomme P, McRea S, Wood TM & Claeyssens M (1988) Chromatographic separation of cellulolytic enzymes. Methods Enzymol 160, 187–193. Pace CN, Vajdos F, Fee L, Grimsley G & Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4, 2411–2423. Chen BL, Baase WA, Nicholson H & Schellman JA (1992) Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C. Biochemistry 31, 1464–1476. Wood TM (1988) Preparation of crystalline, amorphous and dyed cellulase substrates. Methods Enzymol 160, 19–25. Van Tilbeurgh H, Loontiens FG, De Bruyne CK & Claeyssens M (1998) Fluorogenic and chromogenic glycosides as substrates and ligands of carbohydrases. Methods Enzymol 160, 45–59. Mopper K & Gindler EM (1973) A new noncorrosive dye reagent for automatic sugar chromatography. Anal Biochem 56, 440–452. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK & Olson AJ (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19, 1639– 1662. Allinger NL (1997) MM2, A hydrocarbon force field utilizing m1 and m2 torsional terms. J Am Chem Soc 99, 8127–8134. Burkert U & Allinger NL (1982) Molecular Mechanics. ACS Monograph 177. American Chemical Society, Washington, DC. FEBS Journal 274 (2007) 356–363 ª 2006 Genencor International Inc. Journal compilation ª 2006 FEBS 363