FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2001263731> ?p ?o ?g. }

• W2001263731 endingPage "11028" @default.
• W2001263731 startingPage "11020" @default.
• W2001263731 abstract "The catalytic domain of XynCDBFV, a glycoside hydrolase family 11 (GH11) xylanase from ruminal fungus Neocallimastix patriciarum previously engineered to exhibit higher specific activity and broader pH adaptability, holds great potential in commercial applications. Here, the crystal structures of XynCDBFV and its complex with substrate were determined to 1.27–1.43 Å resolution. These structures revealed a typical GH11 β-jelly-roll fold and detailed interaction networks between the enzyme and ligands. Notably, an extended N-terminal region (NTR) consisting of 11 amino acids was identified in the XynCDBFV structure, which is found unique among GH11 xylanases. The NTR is attached to the catalytic core by hydrogen bonds and stacking forces along with a disulfide bond between Cys-4 and Cys-172. Interestingly, the NTR deletion mutant retained 61.5% and 19.5% enzymatic activity at 55 °C and 75 °C, respectively, compared with the wild-type enzyme, whereas the C4A/C172A mutant showed 86.8% and 23.3% activity. These results suggest that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-172 disulfide bond is critical to the NTR-mediated interactions. Furthermore, we also demonstrated that Pichia pastoris produces XynCDBFV with higher catalytic activity at higher temperature than Escherichia coli, in which incorrect NTR folding and inefficient disulfide bond formation might have occurred. In conclusion, these structural and functional analyses of the industrially favored XynCDBFV provide a molecular basis of NTR contribution to its thermostability.Background: Thermophilic xylanases are valuable in many industrial applications.Results: The structures of a xylanase XynCDBFV and its complex with xylooligosaccharides were determined, and its N-terminal region (NTR) contributes to thermostability.Conclusion: NTR may stabilize the overall protein folding of XynCDBFV.Significance: The structural and functional investigation of unprecedented NTR of XynCDBFV provides a new insight into the molecular basis of thermophilic xylanases. The catalytic domain of XynCDBFV, a glycoside hydrolase family 11 (GH11) xylanase from ruminal fungus Neocallimastix patriciarum previously engineered to exhibit higher specific activity and broader pH adaptability, holds great potential in commercial applications. Here, the crystal structures of XynCDBFV and its complex with substrate were determined to 1.27–1.43 Å resolution. These structures revealed a typical GH11 β-jelly-roll fold and detailed interaction networks between the enzyme and ligands. Notably, an extended N-terminal region (NTR) consisting of 11 amino acids was identified in the XynCDBFV structure, which is found unique among GH11 xylanases. The NTR is attached to the catalytic core by hydrogen bonds and stacking forces along with a disulfide bond between Cys-4 and Cys-172. Interestingly, the NTR deletion mutant retained 61.5% and 19.5% enzymatic activity at 55 °C and 75 °C, respectively, compared with the wild-type enzyme, whereas the C4A/C172A mutant showed 86.8% and 23.3% activity. These results suggest that NTR plays a role in XynCDBFV thermostability, and the Cys-4/Cys-172 disulfide bond is critical to the NTR-mediated interactions. Furthermore, we also demonstrated that Pichia pastoris produces XynCDBFV with higher catalytic activity at higher temperature than Escherichia coli, in which incorrect NTR folding and inefficient disulfide bond formation might have occurred. In conclusion, these structural and functional analyses of the industrially favored XynCDBFV provide a molecular basis of NTR contribution to its thermostability. Background: Thermophilic xylanases are valuable in many industrial applications. Results: The structures of a xylanase XynCDBFV and its complex with xylooligosaccharides were determined, and its N-terminal region (NTR) contributes to thermostability. Conclusion: NTR may stabilize the overall protein folding of XynCDBFV. Significance: The structural and functional investigation of unprecedented NTR of XynCDBFV provides a new insight into the molecular basis of thermophilic xylanases. Xylans are the major hemicellulose components in the plant cell wall and account for nearly one-third of all renewable organic carbon source on earth (1.Collins T. Gerday C. Feller G. et al.Xylanases, xylanase families, and extremophilic xylanases.FEMS Microbiol. Rev. 2005; 29: 3-23Crossref PubMed Scopus (1300) Google Scholar, 2.Scheller H.V. Ulvskov P. et al.Hemicelluloses.Annu. Rev. Plant Biol. 2010; 61: 263-289Crossref PubMed Scopus (1830) Google Scholar). Xylans are heteropolysaccharides composed of β-1,4-glycosidic bond-linked xylose units as a backbone chain that is usually decorated by different side groups such as methyl group, acetyl group, and other sugar molecules (1.Collins T. Gerday C. Feller G. et al.Xylanases, xylanase families, and extremophilic xylanases.FEMS Microbiol. Rev. 2005; 29: 3-23Crossref PubMed Scopus (1300) Google Scholar, 3.Subramaniyan S. Prema P. et al.Biotechnology of microbial xylanases. Enzymology, molecular biology, and application.Crit. Rev. Biotechnol. 2002; 22: 33-64Crossref PubMed Scopus (518) Google Scholar). Due to its structural complexity, a set of enzymes is required for complete xylan decomposition, including endo-1,4-β-xylanase, β-xylosidase, acetylxylan esterase, arabinase, and α-glucuronidase (3.Subramaniyan S. Prema P. et al.Biotechnology of microbial xylanases. Enzymology, molecular biology, and application.Crit. Rev. Biotechnol. 2002; 22: 33-64Crossref PubMed Scopus (518) Google Scholar, 4.Khandeparker R. Numan M.T. et al.Bifunctional xylanases and their potential use in biotechnology.J. Ind. Microbiol. Biotechnol. 2008; 35: 635-644Crossref PubMed Scopus (102) Google Scholar). Among them, the glycoside hydrolase endo-1,4-xylanase (xylanase, EC 3.2.1.8) is the key enzyme that catalyzes random hydrolysis of the xylan backbone to small fragments by cleaving the β-1,4-glycosidic bonds (1.Collins T. Gerday C. Feller G. et al.Xylanases, xylanase families, and extremophilic xylanases.FEMS Microbiol. Rev. 2005; 29: 3-23Crossref PubMed Scopus (1300) Google Scholar). Xylanases have been widely applied in many industries such as feed manufacture, paper and pulp processing, and food industry (5.Viikari L. Jorma Sundquist A.K. Linko M. et al.Xylanases in bleaching. From an idea to the industry.FEMS Microbiol. Rev. 1994; 13: 335-350Crossref Scopus (599) Google Scholar, 6.Polizeli M.L. Rizzatti A.C. Monti R. Terenzi H.F. Jorge J.A. Amorim D.S. et al.Xylanases from fungi. Properties and industrial applications.Appl. Microbiol. Biotechnol. 2005; 67: 577-591Crossref PubMed Scopus (1003) Google Scholar7.Ramalingam A.D.H.a. C. et al.Xylanases and its application in food industry. A review.J. Exp. Sci. 2010; 1: 1-11Google Scholar). These biotechnological treatments usually involve harsh conditions and demand enzymes with good thermostability, broad pH adaptability, and high specific activity. Therefore, numerous research projects have been carried out in search of novel xylanases with favorable properties and to improve performance of available enzymes via direct evolution or rational design approaches. To date, various xylanases have been identified from bacteria, fungi, yeasts, plants, and insects (8.Paës G. Berrin J.G. Beaugrand J. et al.GH11 xylanases. Structure/function/properties relationships and applications.Biotechnol. Adv. 2012; 30: 564-592Crossref PubMed Scopus (293) Google Scholar) and are classified into the glycoside hydrolase (GH) 3The abbreviations used are: GHglycoside hydrolaser.m.s.d.root mean square deviationNTRN-terminal regionE-XynCDBFVE. coli-expressed XynCDBFV; P-. pastoris-expressed XynCDBFVXTIxylotriose. family 5, 8, 10, 11, 30, and 43 according to their protein sequence similarities by CAZy database, with a majority belonging to GH10 and GH11. In terms of biotechnological applications, GH11 xylanases have drawn much attention due to their small sizes, high substrate selectivities, and wide ranges of pH and temperature adaptability (9.Biely P. Vrsanská M. Tenkanen M. Kluepfel D. et al.Endo-β-1,4-xylanase families. Differences in catalytic properties.J. Biotechnol. 1997; 57: 151-166Crossref PubMed Scopus (492) Google Scholar). To date nearly a thousand GH11 xylanases have been characterized from various species, but only 26 structures from bacteria and fungi have been solved (CAZy database). Among them, several are thermophilic/thermostable enzymes, including Dictyoglomus thermophilum XynB (optimal temperature 75 °C) (10.McCarthy A.A. Morris D.D. Bergquist P.L. Baker E.N. et al.Structure of XynB, a highly thermostable β-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 Å resolution.Acta Crystallogr. D Biol. Crystallogr. 2000; 56: 1367-1375Crossref PubMed Scopus (51) Google Scholar), Thermobifida fusca NTU22 Xyl11 (optimal temperature 70 °C) (11.van Bueren A.L. Otani S. Friis E.P. Wilson K.S. Davies G.J. et al.Three-dimensional structure of a thermophilic family GH11 xylanase from Thermobifida fusca.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2012; 68: 141-144Crossref PubMed Scopus (12) Google Scholar), and Thermopolyspora flexuosa XynA (optimal temperature 80 °C) (12.Gruber K. Klintschar G. Hayn M. Schlacher A. Steiner W. Kratky C. et al.Thermophilic xylanase from Thermomyces lanuginosus. High-resolution x-ray structure and modeling studies.Biochemistry. 1998; 37: 13475-13485Crossref PubMed Scopus (133) Google Scholar) from bacteria and Chaetomium thermophilum Xyn11A (optimal temperature 80 °C) (13.Hakulinen N. Turunen O. Jänis J. Leisola M. Rouvinen J. et al.Three-dimensional structures of thermophilic β-1,4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa. Comparison of twelve xylanases in relation to their thermal stability.Eur. J. Biochem. 2003; 270: 1399-1412Crossref PubMed Scopus (179) Google Scholar) from fungus. glycoside hydrolase root mean square deviation N-terminal region E. coli-expressed XynCDBFV; P-. pastoris-expressed XynCDBFV xylotriose. Previously, a GH11 xylanase from an anaerobic ruminal fungus, Neocallimastix patriciarum, was isolated and characterized (14.Lee J.M. Hu Y. Zhu H. Cheng K.J. Krell P.J. Forsberg C.W. et al.Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli.Can J. Microbiol. 1993; 39: 134-139Crossref PubMed Scopus (23) Google Scholar, 15.Liu J.H. Selinger B.L. Tsai C.F. Cheng K.J. et al.Characterization of a Neocallimastix patriciarum xylanase gene and its product.Can J. Microbiol. 1999; 45: 970-974Crossref PubMed Scopus (21) Google Scholar). The catalytic domain of the enzyme (Xyn-CD) exerts optimal activity at pH 6.0, and an alkalophilic mutant XynCDBFV with seven amino acid substitutions was later created via direct evolution using error-prone PCR (16.Chen Y.L. Tang T.Y. Cheng K.J. et al.Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase.Can J. Microbiol. 2001; 47: 1088-1094Crossref PubMed Google Scholar). The recombinant XynCDBFV protein is among the highest active xylanases and possesses an optimal temperature of 65 °C, broad pH adaptability, and remarkable tolerance at pH 10.0 when expressed in E. coli. Therefore, the XynCDBFV is an attractive candidate to be developed as an industrial product. In the present study the recombinant XynCDBFV was expressed in the industrial strain P. pastoris and crystallized. The overall protein fold and ligand complex structure are analyzed in detail. Based on these data, potential factors contributing to the enzyme thermostability are proposed. The synthesized gene encoding XynCDBFV, an engineered mutant of Xyn-CD from N. patriciarum (GenBankTM accession number AF123252) (16.Chen Y.L. Tang T.Y. Cheng K.J. et al.Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase.Can J. Microbiol. 2001; 47: 1088-1094Crossref PubMed Google Scholar), was amplified by using PCR with a forward primer of 5′-CCCGAATTCCAAAGTTTCTGTAGTTCAGCTTCT-3′ and a reverse primer of 5′-CCCGCGGCCGCTTAATCACCAATGTAAACCTTTGCGTA-3′. The gene was then cloned into the vector pPICZαA for the P. pastoris system by EcoRI and NotI to yield pPICZαA/xynCDBFV. The substituted mutants including E109A, C4A, C172A, and C4A/C172A were prepared by using the QuikChange site-directed mutagenesis kit (Agilent) with pPICZαA/xynCDBFV as the template. The genes encoding the deleted mutants of Δ6 (deletion of Gln-1–Ser-6) and Δ11 (deletion of Gln-1–Gly-11) were generated by PCR with full-length xynCDBFV gene as the template. These truncated genes were then cloned into the vector pPICZαA by using EcoRI and NotI to yield pPICZαA/xynCDBFV-Δ6 and pPICZαA/xynCDBFV-Δ11. The sequences of the mutated primers are listed in the supplemental Table S1. Alternatively, the xynCDBFV gene was amplified by using PCR and cloned into the vector pET32 Xa/LIC for E. coli expression system. This vector has designed a His tag before the N terminus of targeted gene for purification purpose. The specific primers used here were 5′-GGTATTGAGGGTCGCCAAAGTTTCTGTAGTTCAGCT-3′ (forward) and 5′-AGAGGAGAGTTAGAGCCTTAATCACCAATGTAAACCTTTGC-3′ (reverse). These above plasmids, except pET32 Xa/LIC-xynCDBFV, were linearized by PmeI and then individually transformed into X33 strain of P. pastoris by electroporation. The transformants were selected on the YPD (yeast extract peptone dextrose) plates containing 100 μg/ml zeocin (Invitrogen). The selected clones were inoculated and amplified in 50 ml of buffered glycerol-complex medium (BMGY; 1% yeast extract, 2% peptone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base (YNB) with ammonium sulfate without amino acids, 4× 10–5% biotin, and 1% glycerol) at 30 °C for 1 day. Then the culture medium of cultured cells was replaced by 20 ml of buffered methanol-complex medium (BMMY; 1% yeast extract, 2% peptone, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogen base (YNB) with ammonium sulfate without amino acids, 4× 10–5% biotin, and 0.5% methanol) to induce protein expression. For protein purification, the supernatants were collected and dialyzed twice against the buffer containing 25 mm Tris, pH 7.5. In addition, the proteins were treated by endoglycosidase H (New England Biolabs) during the dialysis procedure. The proteins were then purified by FPLC system using diethylaminoethyl (DEAE) column (GE Healthcare) and eluted using a linear gradient of 0–250 mm NaCl in the buffer containing 25 mm Tris, pH 7.5. The purified proteins were finally concentrated to 10 mg/ml in 25 mm Tris, pH 7.5, 150 mm NaCl by using Amicon Ultra-15 Centrifugal Filter Units (Millipore), and the purity (>95%) was checked by SDS-PAGE. On the other hand the pET32 Xa/LIC-xynCDBFV plasmid was transformed into BL21 (DE3) strain of E. coli. Then the transformed cells were propagated and induced by adding isopropyl 1-thio-β-d-galactopyranoside for protein expression. The protein was purified by FPLC system using a nickel nitriloacetic acid column. The His-tagged protein was eluted using a gradient of 0–250 mm imidazole in the buffer containing 25 mm Tris, pH 7.5, 150 mm NaCl. The eluted protein was then dialyzed against the buffer containing 25 mm Tris, pH 7.5, 150 mm NaCl, with Factor Xa enzyme to remove the His tag. The mixture was loaded onto another nickel nitriloacetic acid column, and the untagged protein was eluted in the buffer without imidazole for purification of untagged protein. The eluted protein was then dialyzed against the buffer containing 25 mm Tris, pH 7.5, for the DEAE column. Next, the purified protein was eluted using a gradient of 0–250 mm NaCl in the 25 mm Tris, pH 7.5, for the DEAE column. The purified proteins were finally concentrated to 10 mg/ml in 25 mm Tris, pH 7.5, 150 mm NaCl, and the purity was checked by SDS-PAGE. The XynCDBFV and E109A proteins were crystallized by using sitting-drop vapor diffusion method and Crystal Screen kit (Hampton Research). The XynCDBFV protein crystals were obtained from the reservoir solution containing 0.1 m Tris, pH 8.5, and 2 m ammonium sulfate at room temperature for 1 day. The E109A protein crystals were obtained from the reservoir solution containing 0.1 m sodium cacodylate, pH 6.5, 0.2 m ammonium sulfate, 26% PEG8000, and 5% glycerol at room temperature for 2 days. The hexagonal E109A-xylotriose-bound crystal was prepared by soaking with 10 mm xylotriose (Megazyme) in the reservoir solution for 1 h. The cryoprotectants for XynCDBFV and E109A crystals contained 0.1 m Tris, pH 8.5, 2 m ammonium sulfate and 10% glycerol and 0.12 m sodium cacodylate, pH 6.5, 0.24 m ammonium sulfate, 31% PEG8000, and 5% glycerol, respectively. All of the x-ray diffraction data were collected at beam line BL13B1 of National Synchrotron Radiation Research Center in Hsinchu, Taiwan. The diffraction images were processed by using HKL2000 (17.Otwinowski Z. Minor W. et al.Processing of X-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The crystal structure of XynCDBFV was solved by the molecular replacement method with the program Phaser (18.McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. et al.Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14440) Google Scholar) from the CCP4 suite (19.Winn M.D. Ballard C.C. Cowtan K.D. Dodson E.J. Emsley P. Evans P.R. Keegan R.M. Krissinel E.B. Leslie A.G. McCoy A. McNicholas S.J. Murshudov G.N. Pannu N.S. Potterton E.A. Powell H.R. Read R.J. Vagin A. Wilson K.S. et al.Overview of the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 235-242Crossref PubMed Scopus (9205) Google Scholar) using the hypothetical XynCDBFV model generated from the structure of Bacillus subtilis B230 Xyn11X (PDB code 1IGO; 47% sequence identity with XynCDBFV) by the SWISS-MODEL website (20.Arnold K. Bordoli L. Kopp J. Schwede T. et al.The SWISS-MODEL workspace. A web-based environment for protein structure homology modelling.Bioinformatics. 2006; 22: 195-201Crossref PubMed Scopus (6025) Google Scholar, 21.Kiefer F. Arnold K. Künzli M. Bordoli L. Schwede T. et al.The SWISS-MODEL Repository and associated resources.Nucleic Acids Res. 2009; 37: D387-D392Crossref PubMed Scopus (1613) Google Scholar) as a search model. Subsequent model building and structural refinement were carried out by using the programs COOT (22.Emsley P. Cowtan K. et al.Coot. Model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar) and REFMAC5 (23.Murshudov G.N. Skubák P. Lebedev A.A. Pannu N.S. Steiner R.A. Nicholls R.A. Winn M.D. Long F. Vagin A.A. et al.REFMAC5 for the refinement of macromolecular crystal structures.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 355-367Crossref PubMed Scopus (5949) Google Scholar), respectively. The complex structure of E109A-xylotriose was determined by the molecular replacement method with Phaser using refined XynCDBFV structure as a search model. The structural refinements were finished by the programs COOT (22.Emsley P. Cowtan K. et al.Coot. Model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar) and REFMAC5 (23.Murshudov G.N. Skubák P. Lebedev A.A. Pannu N.S. Steiner R.A. Nicholls R.A. Winn M.D. Long F. Vagin A.A. et al.REFMAC5 for the refinement of macromolecular crystal structures.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 355-367Crossref PubMed Scopus (5949) Google Scholar). Some data collection and statistics are summarized in Table 1. All of the structural diagrams were drawn by using PyMOL (24.DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar).TABLE 1Data collection and refinement statistics for the XynCDBFVXynCDBFVE109AE109A-XTIData collectionWavelength (Å)1.000001.000001.00000Resolution (Å)25.00-1.27 (1.32-1.27)25.00-1.32 (1.37-1.32)25.00-1.43 (1.48-1.43)Space groupP64P64P64Unit-cell a, b, c (Å)92.8, 92.8, 42.992.9, 92.9, 43.193.2, 93.2, 43.4No. of unique reflections55625 (5510)50174 (5000)39995 (3947)Redundancy8.8 (8.7)9.1 (8.9)9.1 (9.1)Completeness (%)99.8 (99.9)100.0 (100.0)99.9 (100.0)Mean I/σ(I)49.4 (7.9)44.8 (3.1)37.8 (3.1)Rmerge (%)5.0 (20.4)4.3 (46.7)5.2 (49.2)RefinementNo. of reflections52832 (4096)47540 (3646)37972 (2875)Rwork (95% of data)13.9 (14.7)14.7 (21.2)12.8 (19.1)Rfree (5% of data)16.6 (17.1)18.3 (25.4)16.0 (23.4)r.m.s.d. bonds (Å)0.0130.0140.013r.m.s.d. angles (°)1.651.741.64Ramachandran plot (%)Most favored (%)98.799.198.7Allowed (%)1.30.91.3Disallowed (%)0.00.00.0Mean B (Å2) /atomsProtein10.8/174712.0/173815.6/1743Water30.9/33229.6/35135.7/273Ion19.3/5Ligand26.5/47PDB ID code3WP43WP53WP6 Open table in a new tab The detection of xylanase activity was based on the determination of reducing sugar by using dinitrosalicylic acid method (25.Miller G.L. et al.Use of dinitrosaiicyiic acid reagent for determination of reducing sugar.Anal. Chem. 1959; 31: 426-428Crossref Scopus (22217) Google Scholar, 26.König J. Grasser R. Pikor H. Vogel K. et al.Determination of xylanase, β-glucanase, and cellulase activity.Anal. Bioanal. Chem. 2002; 374: 80-87Crossref PubMed Scopus (89) Google Scholar). In general, the protein solution in 50 mm sodium acetate buffer, pH 5.3, with a proper concentration was mixed with 1% xylan substrate (beechwood, Sigma) in the proportion of 1 to 9 and then incubated at 55 °C for 10 min. The reaction was stopped by adding 1% dinitrosalicylic acid solution and incubated at 100 °C in boiling water for 10 min. The absorbance of A540 nm was measured for calculation of the enzyme activity. The standard curve for calibrating the enzyme activity was determined by 0–0.6 mg/ml xylose solutions. One unit of activity is defined as the amount of enzyme that releases 1 μmol of reducing sugar equivalent to xylose per minute. The crystal structure of XynCDBFV was solved to a resolution of 1.27 Å by molecular replacement using the Xyn11X from B. subtilis B230 as a search model. The data collection and refinement statistics of the wild type, E109A mutant (<0.2% activity, data not shown), and its complex with xylotriose (XTI, E109A-XTI) structures are listed in Table 1. The root mean square deviations (r.m.s.d.) of Cα atoms between XynCDBFV and the E109A mutant is 0.374 Å, indicating that the mutation did not cause a significant alteration in the protein structure. To investigate whether the ligand binding could cause significant conformational change, the ligand-free (XynCDBFV and E109A mutant) and ligand-bound (E109A-XTI) structures were superimposed. The r.m.s.d. between these structures ranged from 0.192 to 0.436 Å for all Cα atoms, suggesting that the ligand binding did not result in significant conformational change. As shown in Fig. 1A, the protein structure of XynCDBFV displays a β-jelly-roll fold, which is typical of GH11 family enzymes. It comprises two anti-parallel β-sheets, linked by interconnecting loops and α-helices. The topology of GH11 enzymes is also described as the shape of a right hand (27.Törrönen A. Harkki A. Rouvinen J. et al.Three-dimensional structure of endo-1,4-β-xylanase II from Trichoderma reesei. Two conformational states in the active site.EMBO J. 1994; 13: 2493-2501Crossref PubMed Scopus (262) Google Scholar). The two β-sheets mimic the palm and fingers, whereas the loop β12-β13 is like a thumb. As shown in Fig. 1B, there are two disulfide bridges in the XynCDBFV structure, including DS1 (Cys-4–Cys-172), which connects the N-terminal α1 helix to the strand β14, and DS2 (Cys-50–Cys-60), which joins the strand β5 to the strand β6. The tunnel-like active site cleft (Fig. 1C) is formed by the curved inner β-sheet consisting of β2, β3, β6, β15, β8, β9, β10, β12, and β13 strands (Fig. 1, A and C). The catalytic residues Glu-109 and Glu-202 predicted from sequence alignment of GH11 xylanases were found embedded in this region and located on the strand β10 and β15, respectively (Fig. 1, A and D). The electron density maps in the tunnel-like cleft of the E109A-XTI structure clearly indicated the presence of three β-1,4-linked xylosyl moieties in the +1 to +3 subsites and two in the −2 to −3 subsites (Fig. 2A). The sugar molecules were modeled into the wild-type XynCDBFV to display the detailed interactions between the substrates and the surrounding residues (Fig. 2B). Trp-32 and Trp-125 provide stacking forces to sugar units in the subsites −2 and +3, respectively. Trp-32 further forms a hydrogen bond to the hydroxyl group of the −3 sugar. Glu-30, Arg-61, and Tyr-100 form hydrogen bonds to the −2 sugar unit. Asp-57, Tyr-94, Tyr-111, Arg-148, Gln-161, and Glu-202 (one of catalytic residues) form hydrogen bonds to sugar moiety in the +1 subsite. Arg-148 also forms a hydrogen bond to the +2 sugar unit. The −1 sugar moiety was not observed from our crystal structure, but the subsite can be predicted from the superimposition of the XynCDBFV-XTI model and the complex structure of XynII from Trichoderma reesei (PDB code 4HK9) (28.Wan Q. Zhang Q. Hamilton-Brehm S. Weiss K. Mustyakimov M. Coates L. Langan P. Graham D. Kovalevsky A. et al.X-ray crystallographic studies of family 11 xylanase Michaelis and product complexes. Implications for the catalytic mechanism.Acta Crystallogr. D Biol. Crystallogr. 2014; 70: 11-23Crossref PubMed Scopus (29) Google Scholar). The superimposition indicates that several residues might participate in the −1 subsite formation including Asp-57, Arg-148, Pro-151, and Gln-161 along with two catalytic residues, Glu-109 and Glu-202 (Fig. 2C). Among these ligand-interacting residues, Trp-32, Tyr-100, Tyr-111, Gln-161, and Pro-151 are strictly conserved in GH11 xylanase, indicating their vital roles in catalytic reactions. From protein sequence alignment and structure superimposition, we found that XynCDBFV carries an extended N-terminal region (NTR) that is unique in the GH11 family (Fig. 3, A and B). The NTR is composed of 11 amino acids and spans the convex side of the palm β-sheet, whereas other GH11 enzymes start from the strand β1 or β2 (Fig. 3B). There are several interactions formed between the NTR and the nearby β-strands including β4, β5, β14, β16, and β17 (Fig. 3C). Two stacking interactions are formed between NTR-Phe-3 and β17-Tyr-218 and between NTR-His-9 and β4-Tyr-43 (Fig. 3C). NTR-His-9 is also at a hydrogen bonding distance to β5-Ser-47 and β16-Asp-215. Notably, NTR-Cys-4 forms the DS1 disulfide bond with β14-Cys-172. Through these interactions, the NTR is stably attached to the catalytic core of XynCDBFV instead of hanging freely as a flexible segment. To investigate the functional significance of NTR, we constructed the NTR-truncated mutants, including Δ6 (Gln-1–Ser-6 deletion) and Δ11 (Gln-1–Gly-11 deletion) (Fig. 4A), and analyzed their performance. As shown in Fig. 4B, the enzymatic activities of mutants Δ6 and Δ11 were reduced to 80.9 and 61.5% at 55 °C, respectively, compared with the wild-type protein. Remarkably, the variations between wild-type and mutant enzymes were even larger at higher temperatures. As the wild-type XynCDBFV showed enhanced activity at 65 °C (140%) and 75 °C (151.8%), the relative activities of Δ6 and Δ11 were reduced to 33.7 and 20.7% at 65 °C and 21 and 19.6% at 75 °C (Fig. 4B). These results clearly demonstrated that the NTR plays a role in the catalytic activity of XynCDBFV, and its presence is especially important for the enzyme to exert thermophilic functions. As mentioned, there is a disulfide bond DS1 formed between the NTR and strand β14. To further investigate the role of the DS1 linkage, mutants C4A, C172A, and C4A/C172A were constructed. Similar to the results of the NTR-truncated mutants, the DS1-removed mutants showed lower activities comparing to the wild-type enzyme at 55 °C, and their activities further declined when the tested temperature was increased to 65 °C (39.3–44.8%) and 75 °C (20.8–23.3%) (Fig. 4C). Protein expression levels of all mutants were similar to that of wild type (data not shown). These results suggested that the NTR is important for the catalytic activity and is an essential element for thermophilicity of XynCDBFV, and its function is highly dependent on the presence of disulfide bond DS1. In the beginning we tried to use E. coli-expressed protein (E-XynCDBFV) for structural study because the E. coli expression system is easier to handle than P. pastoris expression system. The E-XynCDBFV was easily expressed and purified but failed to be crystallized despite the protein being catalytically active (Table 2) . When the purified proteins were subjected to SDS-PAGE analysis, the E-XynCDBFV formed a significant amount of dimer and trimer in the absence of reducing agent, suggesting the presence of one or more free Cys residues on the protein surface (Fig. 5) . Reducing agent was supplemented during E-XynCDBFV crystallization to prevent formation of nonspecific disulfide bonds, but no crystal was obtained. However, the crystal structure solved in this study indicated that the four Cys residues in XynCDBFV form two pairs of disulfide bonds, and there is no free thiol group exposed to the bulk solvent. Indeed, the recombinant P-XynCDBFV, which was successfully crystallized, showed much less tendency of dimer formation (Fig. 5).TABLE 2Specific activity of XynCDBFV expressed in E. coli and P. pastorisSpecific activity at pH 5.355 °C65 °C75 °Cunits/mgE. coli4009.7 ± 115.6 (82 ± 2.4%)5747.6 ± 270.2 (117.5 ± 5.5%)5778.3 ± 73.0 (118.2 ± 1.5%)P. pastoris4889.9 ± 218.3 (100 ± 4.5%)7611.6 ± 382.4 (155.7 ± 7.8%)7995.3 ± 178.6 (163.5 ± 3.7%)" @default.
• W2001263731 created "2016-06-24" @default.
• W2001263731 creator A5015001085 @default.
• W2001263731 creator A5017609052 @default.
• W2001263731 creator A5020077494 @default.
• W2001263731 creator A5031194154 @default.
• W2001263731 creator A5036455541 @default.
• W2001263731 creator A5055645197 @default.
• W2001263731 creator A5060032785 @default.
• W2001263731 creator A5065536494 @default.
• W2001263731 date "2014-04-01" @default.
• W2001263731 modified "2023-10-14" @default.
• W2001263731 title "Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum" @default.
• W2001263731 cites W1495442199 @default.
• W2001263731 cites W1539796472 @default.
• W2001263731 cites W154700294 @default.
• W2001263731 cites W1979565029 @default.
• W2001263731 cites W1982370265 @default.
• W2001263731 cites W1982657423 @default.
• W2001263731 cites W1984778603 @default.
• W2001263731 cites W1989552928 @default.
• W2001263731 cites W1991253239 @default.
• W2001263731 cites W1994470547 @default.
• W2001263731 cites W2000028565 @default.
• W2001263731 cites W2003139549 @default.
• W2001263731 cites W2009674410 @default.
• W2001263731 cites W2010670537 @default.
• W2001263731 cites W2011374678 @default.
• W2001263731 cites W2027445263 @default.
• W2001263731 cites W2031401902 @default.
• W2001263731 cites W2049731598 @default.
• W2001263731 cites W2057318144 @default.
• W2001263731 cites W2071945275 @default.
• W2001263731 cites W2086012721 @default.
• W2001263731 cites W2086356567 @default.
• W2001263731 cites W2086924109 @default.
• W2001263731 cites W2094797486 @default.
• W2001263731 cites W2097382368 @default.
• W2001263731 cites W2102687988 @default.
• W2001263731 cites W2108921801 @default.
• W2001263731 cites W2116466817 @default.
• W2001263731 cites W2118200955 @default.
• W2001263731 cites W2132242249 @default.
• W2001263731 cites W2138022937 @default.
• W2001263731 cites W2141629064 @default.
• W2001263731 cites W2142111674 @default.
• W2001263731 cites W2144081223 @default.
• W2001263731 cites W2147012166 @default.
• W2001263731 cites W2152301430 @default.
• W2001263731 cites W2152756549 @default.
• W2001263731 cites W2159211495 @default.
• W2001263731 cites W2163341755 @default.
• W2001263731 cites W2164155599 @default.
• W2001263731 cites W2168162542 @default.
• W2001263731 cites W2168675755 @default.
• W2001263731 cites W4246533287 @default.
• W2001263731 cites W4253164051 @default.
• W2001263731 doi "https://doi.org/10.1074/jbc.m114.550905" @default.
• W2001263731 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4036243" @default.
• W2001263731 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24619408" @default.
• W2001263731 hasPublicationYear "2014" @default.
• W2001263731 type Work @default.
• W2001263731 sameAs 2001263731 @default.
• W2001263731 citedByCount "61" @default.
• W2001263731 countsByYear W20012637312014 @default.
• W2001263731 countsByYear W20012637312015 @default.
• W2001263731 countsByYear W20012637312016 @default.
• W2001263731 countsByYear W20012637312017 @default.
• W2001263731 countsByYear W20012637312018 @default.
• W2001263731 countsByYear W20012637312019 @default.
• W2001263731 countsByYear W20012637312020 @default.
• W2001263731 countsByYear W20012637312021 @default.
• W2001263731 countsByYear W20012637312022 @default.
• W2001263731 countsByYear W20012637312023 @default.
• W2001263731 crossrefType "journal-article" @default.
• W2001263731 hasAuthorship W2001263731A5015001085 @default.
• W2001263731 hasAuthorship W2001263731A5017609052 @default.
• W2001263731 hasAuthorship W2001263731A5020077494 @default.
• W2001263731 hasAuthorship W2001263731A5031194154 @default.
• W2001263731 hasAuthorship W2001263731A5036455541 @default.
• W2001263731 hasAuthorship W2001263731A5055645197 @default.
• W2001263731 hasAuthorship W2001263731A5060032785 @default.
• W2001263731 hasAuthorship W2001263731A5065536494 @default.
• W2001263731 hasBestOaLocation W20012637311 @default.
• W2001263731 hasConcept C181199279 @default.
• W2001263731 hasConcept C185592680 @default.
• W2001263731 hasConcept C196032511 @default.
• W2001263731 hasConcept C2775859485 @default.
• W2001263731 hasConcept C2780120296 @default.
• W2001263731 hasConcept C55493867 @default.
• W2001263731 hasConcept C86803240 @default.
• W2001263731 hasConcept C89423630 @default.
• W2001263731 hasConceptScore W2001263731C181199279 @default.
• W2001263731 hasConceptScore W2001263731C185592680 @default.
• W2001263731 hasConceptScore W2001263731C196032511 @default.
• W2001263731 hasConceptScore W2001263731C2775859485 @default.
• W2001263731 hasConceptScore W2001263731C2780120296 @default.
• W2001263731 hasConceptScore W2001263731C55493867 @default.

• next