Paenibacillus curdlanolyticus
Transcription
Paenibacillus curdlanolyticus
Table of Contents The C-terminal region of xylanase domain in Xyn11A from Paenibacillus curdlanolyticus B-6 plays an important role in structural stability Junjarus Sermsathanaswadi & Somsak Pianwanit & Patthra Pason & Rattiya Waeonukul & Chakrit Tachaapaikoon & Khanok Ratanakhanokchai & Krisna Septiningrum & Akihiko Kosugi Received: 7 February 2014 / Revised: 31 March 2014 / Accepted: 2 April 2014 / Published online: 1 May 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases of Paenibacillus species. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the β6- and β12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain. J. Sermsathanaswadi Department of Chemical Technology, Faculty of Science and Technology, Suan Dusit Rajabhat University, 295 Rajasrima Rd., Dusit, Bangkok 10300, Thailand J. Sermsathanaswadi : K. Ratanakhanokchai School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkuntien, Bangkok 10150, Thailand J. Sermsathanaswadi : K. Septiningrum : A. Kosugi Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan Keywords Paenibacillus curdlanolyticus . Xylanase . GH family-11 . CBM . Xylan degradation S. Pianwanit Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Introduction P. Pason : R. Waeonukul : C. Tachaapaikoon Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkuntien, Bangkok 10150, Thailand Plant biomass contains a complex mixture of polysaccharides, such as cellulose, hemicellulose (xylan and galactomannan), pectic substances (galacturonan and arabinogalactan) and other polysaccharides (e.g., type II arabinogalactan and fucoxyloglucan; Caffall and Mohnen 2009; McNeil et al. 1984; Scheller and Ulvskov 2010), and is expected to be utilized as an abundant renewable resource. Therefore, complete and K. Septiningrum : A. Kosugi (*) University of Tsukuba Graduate School of Life and Environmental Sciences, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan e-mail: [email protected] Reproduced from Appl. Microbiol. Biotechnol. 98: 8223–8233 (2014). Khanok Ratanakhanokchai: Participant of the 18th UM, 1990-1991. 213 rapid hydrolysis of these polysaccharides requires not only β1,4-glycosidic chain-cleaving enzymes, such as endo-β-1,4glucanase, cellobiohydrolase and β-glycosidase, but also the cooperation of numerous enzymes such as xylanolytic enzymes and side chain-cleaving enzymes like β-1,4-xylanase and α-L-arabinofuranosidase (Caffall and Mohnen 2009; McNeil et al. 1984). Among these plant cell wall degradable enzymes, β-1,4-xylanases catalyze the hydrolysis of the β1,4-D-xylosidic linkage in xylan, one of most abundant plant cell wall polysaccharides. The enzymatic hydrolysis of xylan has various potential industrial applications in areas such as food engineering, bio-ethanol production, and cellulose pulp production (Pauly and Keegstra 2010; Saha 2003). The β-1,4-xylanases are generally included in glycosyl hydrolase family-10 (GH10) and -11 (GH11) based on primary structure similarity and three-dimensional (3D) structure homology. GH11 xylanases are highly specific, displaying exclusive substrate specificity toward xylose-containing substrates and a preference for insoluble polymeric substrates. The structures of GH11 are highly homologous and contains a single major α-helix and two extended pleated β-sheets which form a jelly-roll fold (Paës et al. 2012). The structural features include a compact globular structure and a thumb-like structure as an 11-residue long loop that connects β-strands β8 and β7, and a long cleft that spans the entire molecule and contains the active site (Paës et al. 2012; Wakarchuk et al. 1994). The catalytic machinery is composed of two glutamate residues, acting as a nucleophile and an acid/base catalyst, located in the middle of the long cleft (Paës et al. 2012). Presently, 874 GH11 xylanase sequences are available in the CAZy database (http://www.cazy.org/Home.html) and 27 structures for GH11 xylanases have been deposited in the Protein Data Bank (PDB; http://www.pdb.org/pdb/home/home.do; Berman et al. 2003; Paës et al. 2012). A facultative anaerobic bacterium Paenibacillus curdlanolyticus B-6 (BCC no. 11175, National Center for Genetic Engineering and Biotechnology Thailand), isolated from an anaerobic digester which was fed pineapple waste, is a true cellulolytic/xylanolytic organism (Pason et al. 2006). Recently, we determined that P. curdlanolyticus B-6 is able to produce an extracellular multienzyme complex composed of a 280 kDa protein, several minor xylanases and cellulases, and major xylanases of about 40 kDa. The major xylanase subunit known as Xyn11A contains two functional domains belonging to GH11 and carbohydrate binding module family-36 (CBM36) and a relatively unique glycine and asparaginerich long linker (linker; Pason et al. 2010). In this study, the roles of the functional domains of Xyn11A for xylan degradation activity were investigated through enzymatic characterization and conformational analysis using recombinant proteins that included truncated linkers and CBM36. We confirmed the important role of a seven amino acid neighbor linker in the xylan degradation activity of Xyn11A. This is first report to confirm the importance of the C-terminal region of the GH11 xylanase catalytic domain for a Paenibacillus species. Materials and methods Bacterial strains and plasmids P. curdlanolyticus B-6 (Pason et al. 2006) was deposited with the BIOTEC Culture Collection of the National Center for Genetic Engineering and Biotechnology (BIOTEC) Thailand, with the accession number BCC no. 11175. P. curdlanolyticus B-6 was grown on Berg’s mineral salt medium at pH 7.0 (Berg et al. 1972; Pason et al. 2010) containing 2 g of sodium nitrate, 0.5 g of K2HPO4, 0.2 g of MgSO4 ·7H2O, 0.02 g of MnSO4 · H2O, 0.02 g of FeSO4 ·7H2O, and 0.02 g of CaCl2 ·2H2O, and supplemented with 5 g of oat spelt xylan (Sigma-Aldrich, St Louis, MO, USA) per liter of distilled water. Chemicals were purchased from Wako Pure Chemical (Osaka, Japan). Escherichia coli JM109 (Takara Bio, Shiga, Japan) and BL21 (DE3), and plasmids pET19b (Merck, Darmstadt, Germany) served as cloning hosts, expression hosts and vector, respectively. E. coli cells were grown at 37 °C in Luria– Bertani (LB) medium containing ampicillin (100 μg/ml). Preparation of recombinant Xyn11A proteins from P. curdlanolyticus B-6 Genomic DNA was prepared by phenol/chloroform extraction (Pason et al. 2010) and plasmid DNA using a QIAprep spin miniprep kit in accordance with the manufacturers’ protocols (Qiagen, Frederick, MD). The oligonucleotide primers used in this research are listed in Table 1. To produce the recombinant proteins Xyn11A, XynXL, XynX, LS11, LS7, LS4, and LS1 in E. coli, expression plasmids pET-XynA, pET-XynXL, pETXynX, pET-LS11, pET-LS7, pET-LS4, and pET-LS1 were prepared using the xyn11A gene (GenBank accession number FJ956758) from P. curdlanolyticus B-6 (Fig. 1a). Polymerase chain reaction (PCR) was performed with Ex Taq polymerase or PrimeSTAR® HS DNA Polymerase (Takara Bio) under standard conditions according to the manufacturer’s instructions. Amplified fragments were inserted between the NdeI and Bpu1102I sites of pET19b. The constructed plasmids were transformed into E. coli competent cells, and positive clones were verified by DNA sequencing. Production and purification of Xyn11A and its derivatives The E. coli BL21(DE3) strains harboring pET-XynA, pETXynXL, pET-XynX, pET-LS7 pET-LS4, pET-LS3, and pETLS1 were grown at 37 °C in 300 ml of LB medium supplemented with ampicillin (100 μg/ml) until the absorbance 214 Table 1 Oligonucleotide primers used for construction of Xyn11A and its derivatives Primer ATTGCTCAGCGCCGCCGATTGTAATCGTAT Mini Profinity IMAC and a Bio-Gel P6 desalting cartridge in accordance with the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). Protein concentrations were determined using the Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA) with bovine serum albumin as the standard. The homogeneity of the purified proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed on 5 to 20 % gradient polyacrylamide gels (Atto, Tokyo, Japan) according to manufacturer’s instructions. Samples used for SDS-PAGE were boiled for 5 min in sample buffer containing dithiothreitol (DTT; Sigma-Aldrich). After electrophoresis, gels were stained with Coomassie brilliant blue R-250 (BioRad Laboratories). Molecular mass standards were from BioRad Laboratories. ATTGCTCAGCTGTAATCGTATTGCTTGTAA TTGTCGCGCT Enzyme activity Sequence 5′ → 3′ Xyn11A XYNW-for* XYNW-rev XynXL GGAATTCCATATGGTA ACGATTACGAAT XL-rev XynX X-rev LS11 LS11-rev LS7 LS7-rev LS4 LS4-rev ATTGCTCAGCACCTGTATTACCGCCGCCA ATTGCTCAGCATTGATTTCCAAATAATCGA ATTGCTCAGCCGCGCTTCCGCTGCTTTGGT ATTGCTCAGCGCCGCCGCCGTTGCC LS1 LS1-rev Xylanase activity was measured by determining the amount of reducing sugar released from birchwood xylan, oat-spelt xylan, and arabinoxylan (Sigma-Aldrich; Okada and Shinmyo 1988). The reaction mixture consisted of 0.9 ml of 0.5 % (w/v) xylan substrate in 0.1 M sodium acetate buffer at pH 6.0 and 0.1 ml enzyme (Pason et al. 2010). After incubation for 10 min, the reaction was stopped by boiling, and the mixture was separated by centrifugation at 12,100×g for 10 min. Released reducing sugars were quantified by the Somogyi– Nelson method with xylose as a standard (Wood and Bhat 1988). Xylanase activity (1 unit) was defined as the amount of enzyme that liberated 1 μmol of reducing sugar in 1 min under the above conditions (Okada and Shinmyo 1988). Endoglucanase, mannanase, and pectinase activities were measured based on the amount of reducing sugar liberated from carboxymethylcellulose (CMC), barley glucan, carob ATTGCTCAGCATTGCTTGTAATTGTCGCGC NdeI and Bpu1102I restriction sites are underlined. * XYNW-for was used for the construction of all forward primers reached 0.6–0.8 at 600 nm. Protein expression was carried out at 18 °C with the addition of 1 mM isopropyl-thio-β-D-galactoside (IPTG) to the culture media. After cultivation for 16 h, E. coli cells were harvested by centrifugation (5,000×g, 10 min, 4 °C) and were frozen at −80 °C for 24 h. The frozen cell pellet was resuspended in 50 mM sodium phosphate buffer (pH 7.4), and recombinant proteins were extracted by sonication. Cell free extracts were separated into lysates and cell debris by centrifugation (12,100×g, 10 min, 4 °C). Each recombinant protein was purified with the Profinia affinity chromatography protein purification system using Bio-Scale Fig. 1 Schematic representation of the molecular architecture of Xyn11A (a) and its truncated derivatives (b). The molecular architecture of each domain designed based on CDD analysis. Capital letters indicate a part of the amino acid sequence of the GH11 domain, the linker region and CBM36. Boxed amino acids indicated highly conserved amino acids of GH11 xylanases of Paenibacillus and Bacillus species Histidine tags of each recombinant protein are indicated as H- in the N-terminal region. Numbers in parentheses show the predicted molecular mass 215 based on Xyn11A. Molecular graphics were prepared using the homology model of the UCSF Chimera program (Beckham et al. 2010), and energy minimization was performed using the AMBER10 software package (Case et al. 2005; Salomon-Ferrer et al. 2013). galactomannan, arabinan, and polygalacturonic acid (all from Sigma-Aldrich), respectively, at a 0.5 % (v/w) final concentration. β-Glucosidase, β-xylosidase, and α-Larabinofuranosidase activities were based on measurement of p-nitrophenol release from p-nitrophenyl β-D-glucoside (PNPG), p-nitrophenyl β-D-xyloside (PNPX), and p-nitrophenyl β-D-arabinopyranoside (PNPA), respectively (all from Sigma-Aldrich; Matsuo and Yasui 1988). The optimal pH was determined with birchwood xylan in buffer at pHs 4.0 to 9.0 at 60 °C. Buffers used were 0.1 M sodium acetate buffer for pH 4.0 to 6.0, 0.1 M phosphate buffer for pH 6.0 to 8.0, and 0.1 M Tris–HCl buffer for pH 8.0 to 9.0. For pH stability, the enzyme was preincubated without substrate in buffers of different pHs for 1 h at 37 °C, and then the xylanolytic activity was measured at 60 °C for 10 min (pH 6.0). The optimal temperature was determined at pH 6.0 (sodium acetate buffer) from 40 to 80 °C. Thermostability was monitored by preincubating the enzyme without substrate in sodium acetate buffer (pH 6.0) for 30 min at 40 to 100 °C. Residual enzyme activity in each case was then assayed under standard assay conditions. For the determination of kinetics parameters, Km and Vmax values for the purified recombinant enzymes were analyzed using the Lineweaver–Burk method. Enzyme activity was assayed for 10 min at 60 °C in sodium acetate buffer (pH 6.0) containing 1 to 10 mg/ml birchwood xylan. Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy was performed on a J-820 spectropolarimeter (Jasco, Easton, MD) using a rectangular quartz cell with a 1 mm path length. Spectra were acquired using a 1-s time response and a 20 nm/min scan speed; the spectra were averaged for four acquisitions. Individual proteins were analyzed at 7.97 μM in MilliQ water. The percentage of beta-pleated sheets were calculated by secondary structural analysis using JWSSE-480 program (Jasco, Easton, MD; Chang et al. 1978). Energy minimization All hydrogen atoms were added to the homology-modeled structure of Xyn11A using the builder module, followed by energy minimization with the AMBER 10 software package (Case et al. 2005; Salomon-Ferrer et al. 2013). The protein then was solvated with ~9100 TIP3P water molecules in a 12 Å cubic box (Jorgensen et al. 1983). Sodium ions were added to neutralize the system. Subsequently, the solvated protein was minimized. First, the steepest descent algorithm was used for 1,000 steps to remove close van der Waals contacts, followed by 2,000 steps of the more efficient conjugate gradient algorithm until a tolerance of 0.01 kcal/mol/Å was reached. After minimization, the layer water was removed. Validation of the model was carried out using a Ramachandran plot calculation computed with the Procheck program (Laskowski et al. 1993). Hydrolysis activity of xylooligosaccharides using Xyn11A and its derivatives Xyn11A, XynXL, and XynX were incubated in sodium acetate buffer (pH 6.0) containing 0.5 % (w/v) xylooligosaccharides (X2–X6; xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose; Megazyme International, Wicklow, Ireland) overnight at 60 °C. After incubation, the reactions were stopped by boiling, and the mixtures were separated by centrifugation at 12,100×g for 10 min. The monodsaccharide and oligosaccharide contents in the supernatants were measured by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) with a refractive index (Shimadzu RID-10A) detector on a Bio-Rad Aminex HPX-87P column (Bio-Rad Laboratories) operated at 80 °C with MilliQ-filtered water (Millipore, Billerica, MA, USA) at a flow rate of 0.4 ml/min. Molecular docking Xylooligosaccharides that are likely to be involved in substrate binding were generated by the SWEET-II program (Bohne et al. 1998, 1999), and energy minimization was performed with HyperChem 8.0 (Hypercube, Gainesville, FL). The program GOLD version 3.0.1 (Verdonk et al. 2003) was used to generate and rank initial potential binding poses for xylooligosaccharides to the protein target. Flexible xylooligosaccharides were docked to the active site of Xyn11A using a Lamarckian Genetic Algorithm. The simulation consisted of 100 docking runs, with 10,000 generations in each run (at a maximum of 1,000,000 energy evaluations). After docking, all structures generated for the single compound were assigned to clusters based on a tolerance of 2 Å for all atom root-mean-square deviations (RMSDs) from the lowest energy structure. Homology modeling ESPript was used for sequence based alignment (http://espript. ibcp.fr/ESPript/ESPript/) (Gouet et al. 1999). Protein structure modeling of a family 11 xylanase from Bacillus sp. 41 m-1 obtained from the SWISS-MODEL protein-modeling server (http://swissmodel.expasy.org/: PDB accession number 2DCJ_A, chain A of alkaliphilic Xynj from Bacillus sp. 41 m-1) was used as the template structure for building the three-dimensional (3D) structure of the recombinant proteins 216 kDa Results M 1 2 3 4 5 6 7 150 100 75 Enzymatic properties of Xyn11A and the role of CBM36 Xyn11A, the 40 kDa major xylanase subunit in the extracellular multienzyme complex of P. curdlanolyticus B-6, was cloned and sequenced previously (Pason et al. 2010). Xyn11A is composed of two major functional domains: a catalytic domain belonging to family-11 glycosyl hydrolases (GH11), and a carbohydrate binding module classified as family-36 (CBM36). A glycine- and asparagine-rich linker was present between the two domains (Fig. 1a). The two functional domains of GH11 and CBM36 were subsequently characterized for their enzymatic functions and protein structures (Boraston et al. 2004; Paës et al. 2012). First, to evaluate the carbohydrate hydrolysis ability of Xyn11A, the enzymatic properties were characterized using recombinant Xyn11A for xylan and other carbohydrate substrates. Xyn11A had a high specific activity for birchwood xylan (89.7±1.2 U/mg protein), oat-spelt xylan (60.5±1.0 U/mg protein) and arabinoxylan (33.6±1.4 U/mg protein), while no activity was observed with PNPG, PNPX, PNPA, carboxymethylcellulose, glucan, arabinan, mannan, or pectin. The pattern of hydrolyzed xylooligosaccharides was analyzed by HPLC. Xyn11A mainly produced xylobiose and xylotriose from xylooligosaccharides, but was not active with xylobiose and was less active with xylotriose than other xylooligosaccharides. The optimal pH for xylanase activity of Xyn11A was 6.0 and the enzyme stability was in the range of pH 6.0 to 7.0. The temperature for maximum activity was found to be 60 °C at pH 6.0. These enzymatic properties and the narrow substrate specificity of Xyn11A correspond to those of other known GH11 xylanases. To measure substrate affinity and catalytic efficiency, the kinetic parameters of Xyn11A toward birchwood xylan were determined by Michaelis– Menten analysis. The initial reaction rates were determined from the kinetic curves of each reaction, which contained different xylan concentrations. The Km and Vmax values for Xyn11A were estimated to be 1.5±0.1 mg/ml and 137.0± 1.0 μmol/min/mg protein, respectively. CBM36 is known to be a calcium-dependent xylan binding module that can form direct interactions with the substrate through a single atom of the bivalent metal (Boraston et al. 2004). To understand the role of CBM36 in Xyn11A, xylan degradation activities were compared with those of XynXL constructed as a recombinant protein with a truncated CBM36 (Figs. 1b and 2). Although there were no differences in xylan degradation activity or other properties between XynXL and Xyn11A when soluble xylan (birchwood xylan) was used as a substrate, the degradation activity of Xyn11A and XynXL for insoluble xylan (oat-spelt xylan) was 31.4±1.7 and 17.9± 2.5 U/mg protein, respectively. These results indicate that CBM36 in Xyn11A helps improve the accessibility to the catalytic domain for an insoluble xylan substrate. 50 37 25 20 Fig. 2 SDS-PAGE analysis of purified Xyn11A and its truncated derivatives. The designed recombinant proteins were produced by Escherichia coli harboring each plasmid (see “Materials and methods” section). Lane M, standard protein molecular mass makers; lane 1, intact Xyn11A; lane 2, XynXL (truncated CBM36); lane 3, XynX (truncated linker and CBM36); lane 4, LS11 (XynX with NTITIGGNGGG of the linker sequence); lane 5, LS7 (XynX with NTITIGG of the linker sequence); lane 6, LS4 (XynX with NTIT of the linker sequence); lane 7, LS1 (XynX with N of the linker sequence). Each lane contains 1 μg of protein Role of the amino acid sequence neighbor linker on the xylanase activity of Xyn11A A glycine- and asparagine-rich linker sequence is located between GH11 and CBM36 in Xyn11A (Fig. 1a). The linker sequence has unique features including a relatively long amino acid sequence that is located from amino acid positions 236 to 268 and consists of 20, 9, and 4 residues of glycine, asparagine, and threonine from a total of 33 amino acids residues, respectively (Fig. 1a). The linker sequence has been frequently observed in several bacterial xylanases belonging to GH11. On the other hand, according to the conserved domain database (CDD; http://www.ncbi.nlm.nih.gov/cdd/), the conserved GH11 xylanase domain included residues 46 to 230 in Xyn11A (Fig 1a). When the amino acid sequence of the N-terminal side neighbor linker sequence of Xyn11A was compared with that of other GH11 xylanases with similar overall structures, it was observed that the 11 amino acid (NTITIGGNGGG) sequence was relatively conserved for other sequences possessing GH11 xylanases of Paenibacillus and Bacillus species (Fig. 3). To determine the role of the 11 amino acids in Xyn11A, XynX, in which the linker sequence including the 11 amino acids was removed from XynXL, was also constructed as a recombinant protein (Figs. 1b and 2). When the xylan hydrolysis activity of XynX was measured using arabinoxylan, birchwood xylan, and oat-spelt xylan as substrates, the degradation activities markedly decreased to almost undetectable levels (<0.3±0.05 U/mg protein). XynX also showed a loss of hydrolysis activity for xylooligosaccharides. Although similar research characterizing xylanase activity using 217 Fig. 3 Amino acid sequence alignment of linker sequences and bacterial GH11 xylanases. The listed xylanases have high homology of over 56 % for GH11 of Paenibacillus curdlanolyticus B-6 Xyn11A (top) from BLAST analysis. Accession numbers are shown in parentheses. Strictly and highly conserved residues are marked with asterisks and periods on the bottom of the sequences derivatives with truncated CBM and linker sequences has been reported for other GH11 xylanases (Kosugi et al. 2002; Morris et al. 1998; Paloheimo et al. 2007), in many cases, the xylanase activity was not influenced by truncation of these domains. Thus, the 11 amino acid neighbor linker sequence of Xyn11A appears to play an important role in the catalytic domain. (Fig. 4). When the secondary structure of XynX was compared with that of XynXL, both alignments exhibited a typical GH11 xylanase structure consisting of 12 β-sheets and one major αhelix (Fig. 4). Although the alignment of XynX exhibited an overall β-jelly roll shape, gaps were observed in the beginning position (Leu100-Ser101) of β6 and the end position (Thr229Leu235) of the β12-sheets compared with XynXL (Fig. 4). On the other hand, to insure the proteins were not improperly folded, CD spectroscopy was performed to compare the secondary structures of XynXL and XynX individually. Deconvolution of the CD spectra indicated that XynXL and XynX have a predominant β-sheet and the ratio of β-sheet exhibits 99 and 91 %, respectively. These results may reflect the gap of β6- and β12sheets with XynX. Although the spectra of XynXL and XynX showed a slightly misfitting and deepening at 220 nm (Fig. 5), the spectrum of XynX was almost the same as XynXL. Thus, the 3D structure of GH11 with or without the linker sequence including the 11 amino acids in Xyn11A XynX truncated by 11 amino acids (NTITIGGNGGG) showed a loss of the ability to degrade xylan for all substrates. Truncation of the conserved amino acid sequences may affect the structure of the GH11 domain. To confirm whether truncation of the 11 amino acids influences GH11 structure, a secondary structural alignment of XynXL and XynX was carried out using ESPript Fig. 4 Sequence-based alignment of the GH11 domain of XynXL and XynX. Secondary structural elements are shown above for the 3D solved structure. Differences in structural elements are highlighted in yellow 218 Mol. ellip. (x10-3 dg.cm2/dmol) 6 5 XynXL 4 XynX org/) based on the X-ray structure of Bacillus sp. 41 m-1 Xynj (PDB accession number 2DCJ, chain A), which showed the highest identity percentage (68 %) among the available amino acid sequences (Fig. 6a). In modeling of XynXL and XynX, significant differences could not be observed between them with or without vector-derived amino acid sequences such as the His-tag. The gaps in the β6- and β12-sheets in XynX were represented as short β-strands in the 3D structure (Fig. 6a). When molecular graphics were generated based on the 3D structures of Xyn-XL and Xyn-X using the UCSF chimera program (Beckham et al. 2010), small grooves were observed in the β-jelly roll hold area near the β6- and β12-sheets, where fingers in the GH 11 structure are observed (Fig. 6b). On the other hand, when the superimposed models were compared using the 3D structures of XynXL and XynX, no differences in the positions and angles of the two active site glutamates (Glu128 and Glu218) located on either side of the long open cleft were noted (Fig. 7). Thus, it appears that the xylanase activity loss upon truncation of XynX was not related to the GH11 active site. 3 2 1 0 -1 200 210 220 230 240 250 -2 -3 -4 Wavelength (nm) Fig. 5 CD spectra of XynXL and XynX. XynXL is shown by the solid line, XynX is shown by the dotted line reduced activity of XynX may be not due to drastic alteration in the structure. These results suggested that a minimum of NTITI in the conserved amino acid sequence is not only a part of the GH11 xylanase domain, but is also important for configuring its β6- and β12-sheets. The three-dimensional (3D) structures of XynXL and XynX were generated using the homology model of the SWISSMODEL protein-modeling server (http://swissmodel.expasy. Fig. 6 Comparison of the GH11 structure of XynXL and XynX. Schematic and surface representation of XynXL and XynX are shown above (a) and below (b), respectively. The αhelices and β-strands of each GH11structure are labeled as α, α1, β6, and β12, respectively. The N and C termini of each GH structure are labeled. Dotted circles indicate conformational differences due to truncation of the linker sequence (a). The small groove is indicated by the black arrow on the surface model of XynX (b) Properties of substrate docking in XynX On the superimposed models of XynXL and XynX, flipping of the side chains of Arg163 (Fig. 7), where the subsite related to substrate binding is located, was observed. To date, the a N N 12 α1 12 6 C 6 C XynXL b 219 XynX α analyzed for XynXL and XynX. Analysis of the docking model using xylohexaose as a model substrate revealed that XynXL has a high binding energy of −8.28 kcal/mol in the open cleft area containing the active site, while XynX showed no binding affinity in that area (Table 2). The arginine side chain of the neighboring active site is known to play an important role in substrate binding at subsite (−1) via strong hydrogen bonds (Cuyvers et al. 2011; Jommuengbout et al. 2009; Sabini et al. 1999; Vandermarliere et al. 2008). On the other hand, XynX was able to bind xylohexaose at the secondary binding site located on the “fingertips” (Cuyvers et al. 2011; Table 2). Thus, the loss of xylanase for XynX may be caused by marked decreases in substrate binding affinity at the subsite due to flipping of the Arg163 side chains, indicating that the conserved 11 amino acids also contribute to the stability of the GH11 structure in Xyn11A. To predict what length of amino acid sequence is necessary to recover structure and activity, docking models were analyzed using several XynX models with different amino acids in the conserved sequence. When four XynX models (Fig. 1b) with the conserved amino acid sequence N (LS1), NTIT (LS4), NTITIGG (LS7), and NTITIGGNGGG (LS11) were docked with xylohexaose, the LS1 and LS4 models were unable to bind the xylohexaose in the open cleft near the active site, whereas LS7 and LS11 were found to bind it in the cleft area (Table 2). In addition to the restitution of substrate binding, superimposed models of XynXL, LS7, and LS11 showed that the flipped side chain of Arg163 in XynX returned to its original angle by addition of a linker sequence of 7 or 11 amino acids. To confirm whether the docking models corresponded to actual xylan hydrolysis ability, LS1, LS4, LS7, and LS11 were constructed as recombinant proteins (Figs. 1b and 2) and assessed for xylanase activity. The activity of LS1 and LS4 remained low (10 and 30 %, Arg163 Glu128 Glu218 N C Fig. 7 Structure-based alignment of the GH11 domains of XynXL and XynX. Superposition of the GH11 domains of XynXL (blue) with XynX (yellow) is shown. Two glutamic acids (Glu128 and Glu218) and arginine (Arg163) located in the substrate binding site were superimposed to confirm angles substrate binding sites of several GH11 xylanases have been determined to be involved in xylan degradation (Cuyvers et al. 2011; Jommuengbout et al. 2009; Vandermarliere et al. 2008; Wakarchuk et al. 1994). To determine whether the xylan binding ability is influenced by truncation of the 11 amino acid sequence, substrate docking and binding energies were Table 2 Comparison of the binding interaction and xylanase activity between XynXL and its derivatives Designed proteina XynXL XynX LS1 LS4 LS7 LS11 -8.28 N.D.c -1.47 -4.5 -7.99 -7.97 100 N.D.c 10 30 94 99 Docking modelb Binding energy b (kcal/mol) Relative xylanase activity (%)d N.D. not detected a Features of designed proteins are shown in Fig. 1 b Docking models and binding energy were obtained with SweetII and HyperChem 8.0 software, respectively, as described in “Materials and methods” section. The xylohexaose molecule was modeled in the energetically highest binding region for each protein structure c Relative activity is given as a percentage of xylanase activity. Values are means of triplicate experiments d Relative activity is given as a percentage of xylanase activity. Values are means of triplicate experiments 220 why flipping of the side chain of Arg163 occurred from deletion of the C-terminal amino acids: flipping of the side chain of Arg163 significantly influences the substrate binding ability to the subsite adjacent to the active site. In GH11 xylanases of Bacillus circulans, Bacillus firmus K-1, Bacillus agaradhaerens, and Streptomyces sp. 38, the side chains of arginine and hydrophobic amino acids, such as proline and tyrosine neighboring the active site, are known to have important roles, such as forming hydrogen bonds between the side chains and hydroxyl groups of the xylose moiety in the subsite (de Lemos Esteves et al. 2004; Jommuengbout et al. 2009; Sabini et al. 1999; Wakarchuk et al. 1994). Similarly, when site direct mutation for the arginine related to subsite -1 was performed in GH11 xylanases of B. circulans, xylanase activity was also significantly decreased compared with the activities after other mutations in the subsite (Wakarchuk et al. 1994). We recently determined that P. curdlanolyticus B-6 is able to produce an extracellular multienzyme complex that was composed of a highly glycosylated 280 kDa protein with xylanase activity, two xylanases of 40 kDa (Xyn11A) and 48 kDa, and 60 and 65 kDa proteins having both xylanase and CMCase activities (Pason et al. 2010). From gene sequence analysis of Xyn11A, we concluded that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin–dockerin interactions known in cellulosomes because of the absence of dockerin-like homology sequences observed in cellulosomal enzyme subunits (Doi and Kosugi 2004; Fontes and Gilbert 2010; Pason et al. 2010). To elucidate the role of each subunit in xylan degradation and complex formation, characterization of the possible 280 kDa scaffolding subunit and Xyn11A should be first clarified through understanding the structures and functions of the two major subunits. Fungal and bacterial glycosyl hydrolases are frequently subjected to post-translational modifications, such as N- and O-glycosylation, and CBMs can also be involved in binding polysaccharides in the bacterial cell wall and in enzymes (Ezer et al. 2008; Montanier et al. 2009). Further research on the structure and functions of these enzyme subunits is necessary to clarify the mechanisms of complex formation. respectively; Table 2) using birchwood xylan as a substrate, while the activities of LS7 and LS11 recovered to 94 and 99 % compared with that of XynXL, respectively. These results were in good agreement with the predictions from the substrate docking model analysis using LS1, LS3, LS4, and LS7, and the NTITIGG sequence was confirmed to be necessary to stabilize the structure of GH11 xylanase. Discussion In this study, the xylan degradation ability of P. curdlanolyticus B-6 Xyn11Awas characterized. The GH11 domain and CBM36 of Xyn11A have high homology with the endo-β-1,4 xylanase from Paenibacillus xylaniclasticus (99 % identity, AFN70714), Paenibacillus sp. Aloe-11 (75 % identity, WP_007429110), Paenibacillus peoriae (75 % identity, WP_010347573), Paenibacillus campinasensis (71 % identity, AGG82434), Gracilibacillus lacisalsi (69 % identity, WP_018932277), and Bacillus sp. YA-335 (68 % identity, CAA41784). It is known that glycoside hydrolases that possess CBMs facilitate attack on natural substrates (Boraston et al. 2004). CBMs are thought to target the enzyme toward specific cell wall regions and maintain it in the proximity of the substrate (Boraston et al. 2004). In fact, XynXL shows decreased hydrolysis activity for insoluble xylan substrates as compared with XynX. The CBM36 from Paenibacillus polymyxa xylanase 43A shows calciumdependent binding of xylan and xylooligosaccharides (JamalTalabani et al. 2004). It is also known that CBM36 has structural similarities with CBM6, which has been demonstrated to have a cellulose-binding function (van Bueren et al. 2005). We also found that the CBM domain of Xyn11A was able to bind xylan as well as soluble and insoluble celluloses, such as carboxymethylcellulose, amorphous cellulose, and microcrystalline cellulose (Sermsathanaswadi 2012), suggesting that Xyn11A is able to show flexible binding for complex plant cell wall polysaccharides. The 11 amino acid (NTITIGGNGGG) neighbor linker was highly conserved in other GH11 xylanases, such as Paenibacillus sp. Aloe11, P. peoriae, P. campinasensis, Bacillus sp., and G. lacisalsi (Fig. 3). In particular, the NTITIGG sequence might play an important role in stabilizing GH11 structures, similar to the case with Xyn11A. In fact, loss of the C-terminal seven amino acid neighbor xylanase domain in Xyn11A resulted in the loss of xylanase activity due to structural destabilization which was provoked by gaps in the β6- and β12-sheets. These β-sheets are located dorsal to the active site (Glu 218) of the GH11 xylanase catalytic domain. Thus, the short stretch may involve a part of the catalytic domain of GH family-11 xylanases. These structural features suggested that at least a NTITIGG sequence including a part of the linker is necessary for structural stabilization of the GH11 catalytic domain of Xyn11A. On the other hand, it is unclear Acknowledgments This work was partly supported by Suan Dusit Rajabhat University, Thailand. 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