FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2910361751> ?p ?o ?g. }

• W2910361751 endingPage "4078" @default.
• W2910361751 startingPage "4065" @default.
• W2910361751 abstract "Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8). Unlike for the well-studied GH30-8 enzymes, the structural and functional characteristics of GH30-7 enzymes remain poorly understood. Here, we report the catalytic properties and three-dimensional structure of GH30-7 xylanase B (Xyn30B) identified from the cellulolytic fungus Talaromyces cellulolyticus. Xyn30B efficiently degraded glucuronoxylan to acidic xylooligosaccharides (XOSs), including an α-1,2-linked 4-O-methyl-d-glucuronosyl substituent (MeGlcA). Rapid analysis with negative-mode electrospray-ionization multistage MS (ESI(−)-MSn) revealed that the structures of the acidic XOS products are the same as those of the hydrolysates (MeGlcA2Xyln, n > 2) obtained with typical glucuronoxylanases. Acidic XOS products were further degraded by Xyn30B, releasing first xylobiose and then xylotetraose and xylohexaose as transglycosylation products. This hydrolase reaction was unique to Xyn30B, and the substrate was cleaved at the xylobiose unit from its nonreducing end, indicating that Xyn30B is a bifunctional enzyme possessing both endo-glucuronoxylanase and exo-xylobiohydrolase activities. The crystal structure of Xyn30B was determined as the first structure of a GH30-7 xylanase at 2.25 Å resolution, revealing that Xyn30B is composed of a pseudo-(α/β)8-catalytic domain, lacking an α6 helix, and a small β-rich domain. This structure and site-directed mutagenesis clarified that Arg46, conserved in GH30-7 glucuronoxylanases, is a critical residue for MeGlcA appendage–dependent xylan degradation. The structural comparison between Xyn30B and the GH30-8 enzymes suggests that Asn93 in the β2–α2 loop is involved in xylobiohydrolase activity. In summary, our findings indicate that Xyn30B is a bifunctional endo- and exo-xylanase. Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8). Unlike for the well-studied GH30-8 enzymes, the structural and functional characteristics of GH30-7 enzymes remain poorly understood. Here, we report the catalytic properties and three-dimensional structure of GH30-7 xylanase B (Xyn30B) identified from the cellulolytic fungus Talaromyces cellulolyticus. Xyn30B efficiently degraded glucuronoxylan to acidic xylooligosaccharides (XOSs), including an α-1,2-linked 4-O-methyl-d-glucuronosyl substituent (MeGlcA). Rapid analysis with negative-mode electrospray-ionization multistage MS (ESI(−)-MSn) revealed that the structures of the acidic XOS products are the same as those of the hydrolysates (MeGlcA2Xyln, n > 2) obtained with typical glucuronoxylanases. Acidic XOS products were further degraded by Xyn30B, releasing first xylobiose and then xylotetraose and xylohexaose as transglycosylation products. This hydrolase reaction was unique to Xyn30B, and the substrate was cleaved at the xylobiose unit from its nonreducing end, indicating that Xyn30B is a bifunctional enzyme possessing both endo-glucuronoxylanase and exo-xylobiohydrolase activities. The crystal structure of Xyn30B was determined as the first structure of a GH30-7 xylanase at 2.25 Å resolution, revealing that Xyn30B is composed of a pseudo-(α/β)8-catalytic domain, lacking an α6 helix, and a small β-rich domain. This structure and site-directed mutagenesis clarified that Arg46, conserved in GH30-7 glucuronoxylanases, is a critical residue for MeGlcA appendage–dependent xylan degradation. The structural comparison between Xyn30B and the GH30-8 enzymes suggests that Asn93 in the β2–α2 loop is involved in xylobiohydrolase activity. In summary, our findings indicate that Xyn30B is a bifunctional endo- and exo-xylanase. Xylan is a major component of plant cell walls and hardwood hemicellulose. It is a heteropolysaccharide consisting of β-1,4-d-xylopyranose polymer as the main chain and some side-chain residues, such as α-l-arabinofuranose and 4-O-methyl-d-glucuronic acid (MeGlcA). 2The abbreviations used are: MeGlcAα-1,2-linked 4-O-methyl-d-glucuronosylGH30-7 and GH30-8subfamily 7 and 8, respectively, of glycoside hydrolase family 30Xyn30BGH30-7 xylanase B from T. cellulolyticusXOSxylooligosaccharideEcXynAXynA from Dickeya chrysanthemiBsXynCXynC from Bacillus subtilisCaXyn30AXyn30A from Clostridium acetobutylicumBR-MeGlcA3Xyl3borohydride-reduced aldotetrauronic acidMeGlcA2Xyl2aldotriuronic acidESIelectrospray ionizationMSnmultistage mass spectrometryXyl2xylobioseXyl3xylotrioseXyl4xylotetraoseXyl5xylopentaoseXyl6xylohexaoseHPAEChigh-performance anion-exchange chromatographyPADpulsed amperometric detectionGHglycoside hydrolaseCAZycarbohydrate active enzymesPDBProtein Data BankDNS3,5-dinitrosalicylic acidBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Bacteria and fungi produce several kinds of endo-β-1,4-xylanases (EC 3.2.1.8) to cleave xylan backbones (1Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42 (24270786): D490-D49510.1093/nar/gkt1178Crossref PubMed Scopus (4115) Google Scholar). These enzymes belong to a variety of glycoside hydrolase (GH) families (families 5, 8, 10, 11, 30, 43, 51, 98, and 141) in the Carbohydrate Active Enzymes (CAZy) database (1Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42 (24270786): D490-D49510.1093/nar/gkt1178Crossref PubMed Scopus (4115) Google Scholar). Glucuronoxylan xylanohydrolases (glucuronoxylanases: EC 3.2.1.136) are classified in the GH30 family (previously in the GH5 family) and degrade the glucuronoxylan main chain at the second glycosidic linkage from the MeGlcA residue toward the reducing end (2St John F.J. Rice J.D. Preston J.F. Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and analysis of its role in depolymerization of glucuronoxylan.J. Bacteriol. 2006; 188 (17028274): 8617-862610.1128/JB.01283-06Crossref PubMed Scopus (99) Google Scholar, 3Vršanská M. Kolenová K. Puchart V. Biely P. Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi.FEBS J. 2007; 274 (17381510): 1666-167710.1111/j.1742-4658.2007.05710.xCrossref PubMed Scopus (76) Google Scholar). They exert no or very low effects on xylan or xylooligosaccharides (XOSs) when the MeGlcA side chain is absent (2St John F.J. Rice J.D. Preston J.F. Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and analysis of its role in depolymerization of glucuronoxylan.J. Bacteriol. 2006; 188 (17028274): 8617-862610.1128/JB.01283-06Crossref PubMed Scopus (99) Google Scholar3Vršanská M. Kolenová K. Puchart V. Biely P. Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi.FEBS J. 2007; 274 (17381510): 1666-167710.1111/j.1742-4658.2007.05710.xCrossref PubMed Scopus (76) Google Scholar, 4Valenzuela S.V. Diaz P. Pastor F.I. Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module.Appl. Environ. Microbiol. 2012; 78 (22447606): 3923-393110.1128/AEM.07932-11Crossref PubMed Scopus (56) Google Scholar, 5Padilha I.Q.M Valenzuela S.V. Grisi T.C.S.L. Díaz P. de Araújo D.A.M Pastor F.I. A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil.Int. Microbiol. 2014; 17 (26419457): 175-184PubMed Google Scholar6St John F.J. Crooks C. Dietrich D. Hurlbert J. Xylanase 30 A from Clostridium thermocellum functions as a glucuronoxylan xylanohydrolase.J. Mol. Catal. B Enzym. 2016; 133: S445-S45110.1016/j.molcatb.2017.03.008Crossref Scopus (16) Google Scholar). The bacterial and fungal enzymes are further classified in the GH30 subfamilies 8 (GH30-8) and 7 (GH30-7), respectively, by phylogenetic analysis based on their amino acid sequences (7St John F.J. González J.M. Pozharski E. Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups.FEBS Lett. 2010; 584 (20932833): 4435-444110.1016/j.febslet.2010.09.051Crossref PubMed Scopus (104) Google Scholar). The GH30 enzymes of Actinobacteria are categorized into both GH30–8 and GH30–7. α-1,2-linked 4-O-methyl-d-glucuronosyl subfamily 7 and 8, respectively, of glycoside hydrolase family 30 GH30-7 xylanase B from T. cellulolyticus xylooligosaccharide XynA from Dickeya chrysanthemi XynC from Bacillus subtilis Xyn30A from Clostridium acetobutylicum borohydride-reduced aldotetrauronic acid aldotriuronic acid electrospray ionization multistage mass spectrometry xylobiose xylotriose xylotetraose xylopentaose xylohexaose high-performance anion-exchange chromatography pulsed amperometric detection glycoside hydrolase carbohydrate active enzymes Protein Data Bank 3,5-dinitrosalicylic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Many studies on bacterial GH30-8 glucuronoxylanases have been performed (2St John F.J. Rice J.D. Preston J.F. Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and analysis of its role in depolymerization of glucuronoxylan.J. Bacteriol. 2006; 188 (17028274): 8617-862610.1128/JB.01283-06Crossref PubMed Scopus (99) Google Scholar3Vršanská M. Kolenová K. Puchart V. Biely P. Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi.FEBS J. 2007; 274 (17381510): 1666-167710.1111/j.1742-4658.2007.05710.xCrossref PubMed Scopus (76) Google Scholar, 4Valenzuela S.V. Diaz P. Pastor F.I. Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module.Appl. Environ. Microbiol. 2012; 78 (22447606): 3923-393110.1128/AEM.07932-11Crossref PubMed Scopus (56) Google Scholar, 5Padilha I.Q.M Valenzuela S.V. Grisi T.C.S.L. Díaz P. de Araújo D.A.M Pastor F.I. A glucuronoxylan-specific xylanase from a new Paenibacillus favisporus strain isolated from tropical soil of Brazil.Int. Microbiol. 2014; 17 (26419457): 175-184PubMed Google Scholar6St John F.J. Crooks C. Dietrich D. Hurlbert J. Xylanase 30 A from Clostridium thermocellum functions as a glucuronoxylan xylanohydrolase.J. Mol. Catal. B Enzym. 2016; 133: S445-S45110.1016/j.molcatb.2017.03.008Crossref Scopus (16) Google Scholar, 8Maehara T. Yagi H. Sato T. Ohnishi-Kameyama M. Fujimoto Z. Kamino K. Kitamura Y. St John F.J. Yaoi K. Kaneko S. GH30 glucuronoxylan-specific xylanase from Streptomyces turgidiscabies C56.Appl. Environ. Microbiol. 2018; 84 (29180367): e01850-e01917PubMed Google Scholar). Mutational analyses of EcXynA from Dickeya chrysanthemi (formerly Erwinia chrysanthemi) and BsXynC from Bacillus subtilis and their structural analyses when complexed with aldouronic acids have revealed that the ionic interaction between a conserved Arg residue (Arg293 of EcXynA and Arg272 of BsXynC) and a carboxyl group on the MeGlcA side chain confers specificity for glucuronoxylan (9Urbániková L. Vršanská M. Mørkeberg Krogh K.B.R Hoff T. Biely P. Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase.FEBS J. 2011; 278 (21501386): 2105-211610.1111/j.1742-4658.2011.08127.xCrossref PubMed Scopus (56) Google Scholar, 10St John F.J. Hurlbert J.C. Rice J.D. Preston J.F. Pozharski E. Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase.J. Mol. Biol. 2011; 407 (21256135): 92-10910.1016/j.jmb.2011.01.010Crossref PubMed Scopus (63) Google Scholar11Šuchová K. Kozmon S. Puchart V. Malovíková A. Hoff T. Mørkeberg Krogh K.B.R Biely P. Glucuronoxylan recognition by GH 30 xylanases: a study with enzyme and substrate variants.Arch. Biochem. Biophys. 2018; 643 (29477770): 42-4910.1016/j.abb.2018.02.014Crossref PubMed Scopus (10) Google Scholar). Several GH30-8 xylanases, including those without the conserved Arg residue, have been reported to degrade both glucuronoxylan and arabinoxylan without exhibiting the MeGlcA appendage dependence (12St John F.J. Dietrich D. Crooks C. Balogun P. de Serrano V. Pozharski E. Smith J.K. Bales E. Hurlbert J.C. A plasmid borne, functionally novel glycoside hydrolase family 30, subfamily 8 endoxylanase from solventogenic Clostridium.Biochem. J. 2018; 475 (29626157): 1533-155110.1042/BCJ20180050Crossref PubMed Scopus (18) Google Scholar, 13St John F.J. Dietrich D. Crooks C. Pozharski E. González J.M. Bales E. Smith K. Hurlbert J.C. A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.Acta Crystallogr. D Biol. Crystallogr. 2014; 70 (25372685): 2950-295810.1107/S1399004714019531Crossref PubMed Scopus (26) Google Scholar). In contrast to bacterial enzymes, there are very few reports on fungal GH30-7 xylanases. Cellulolytic fungi, such as Trichoderma reesei, Myceliophthora thermophila, and Talaromyces cellulolyticus, which are promising enzyme sources for hydrolyzing lignocellulosic biomass (14Karnaouri A. Topakas E. Antonopoulou I. Christakopoulos P. Genomic insights into the fungal lignocellulolytic system of Myceliophthora thermophila.Front. Microbiol. 2014; 5 (24995002): 28110.3389/fmicb.2014.00281Crossref PubMed Scopus (62) Google Scholar, 15Fujii T. Inoue H. Yano S. Sawayama S. Strain improvement for industrial production of lignocellulolytic enzyme by Talaromyces cellulolyticus.in: Fang X. Qu Y. Fungal Cellulolytic Enzymes. Springer, Singapore2018: 58-68Crossref Scopus (7) Google Scholar16Peterson R. Nevalainen H. Trichoderma reesei RUT-C30: thirty years of strain improvement.Microbiology. 2012; 158 (21998163): 58-6810.1099/mic.0.054031-0Crossref PubMed Scopus (343) Google Scholar), encode multiple putative GH30-7 xylanases in their genomes. The GH30-7 xylanases are secreted in cellulosic and xylanosic culture conditions (17Adav S.S. Chao L.T. Sze S.K. Quantitative secretomic analysis of Trichoderma reesei strains reveals enzymatic composition for lignocellulosic biomass degradation.Mol. Cell. Proteomics. 2012; 11 (M111.012419) (22355001)10.1074/mcp.M111.012419Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 18Kolbusz M.A. Di Falco M. Ishmael N. Marqueteau S. Moisan M.C. Baptista C.D.S Powlowski J. Tsang A. Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila.Fungal Genet. Biol. 2014; 72 (24881579): 10-2010.1016/j.fgb.2014.05.006Crossref PubMed Scopus (60) Google Scholar). However, information on their catalytic properties is limited, except for those expressed in T. reesei. This fungus produces two types of GH30-7 xylanases possessing exo-xylanase activity toward the reducing end of xylan (XYN IV) and glucuronoxylanase activity similar to the bacterial GH30-8 enzyme (XYN VI) (19Tenkanen M. Vršanská M. Siika-aho M. Wong D.W. Puchart V. Penttilä M. Saloheimo M. Biely P. Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity.FEBS J. 2013; 280 (23167779): 285-30110.1111/febs.12069Crossref PubMed Scopus (60) Google Scholar, 20Biely P. Puchart V. Stringer M.A. Mørkeberg Krogh K.B.R Trichoderma reesei XYN VI: a novel appendage-dependent eukaryotic glucuronoxylan hydrolase.FEBS J. 2014; 281 (25041335): 3894-390310.1111/febs.12925Crossref PubMed Scopus (40) Google Scholar). The notable difference in the sequences of fungal GH30-7 and GH30-8 xylanases is the absence of an Arg293 counterpart (20Biely P. Puchart V. Stringer M.A. Mørkeberg Krogh K.B.R Trichoderma reesei XYN VI: a novel appendage-dependent eukaryotic glucuronoxylan hydrolase.FEBS J. 2014; 281 (25041335): 3894-390310.1111/febs.12925Crossref PubMed Scopus (40) Google Scholar). Without a three-dimensional structure of GH30-7, it is hard to determine the structural underpinnings of the MeGlcA appendage dependence of XYN VI and structural differences between exo-xylanase and glucuronoxylanase. In a preliminary study, we detected two GH30-7 proteins from T. cellulolyticus, termed Xyn30A (NCBI protein ID GAM43270) and Xyn30B (GAM36763), which were secreted as major proteins in a culture containing birchwood glucuronoxylan (Fig. S1). Xyn30A has been predicted to be a putative exo-xylanase from its relatively high sequence similarity with XYN IV (77%), whereas Xyn30B has remained to be identified due to lack of an appropriate GH30-homologous protein. Here, we report the catalytic properties and crystal structure of Xyn30B. Xyn30B exhibited an obvious, MeGlcA appendage–dependent glucuronoxylanase activity. Moreover, we found that Xyn30B exhibits a novel xylobiohydrolase activity wherein xylobiose (Xyl2) is released from the nonreducing end of β-1,4-xylan and XOS. This unique bifunctional activity and the conserved Arg residue found in Xyn30B are discussed based on the structural comparison between GH30-8 and GH30-7 xylanase. The Xyn30B gene is composed of 1,425 bp without introns in the T. cellulolyticus genome and encodes a protein consisting of 474 amino acid residues. Xyn30B has a relatively high amino acid sequence identity with fungal GH30-7 enzymes, such as XYN IV (38.2%) and XYN VI (42.2%) from T. reesei and XYLD (53.4%) from Bispora sp. (19Tenkanen M. Vršanská M. Siika-aho M. Wong D.W. Puchart V. Penttilä M. Saloheimo M. Biely P. Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity.FEBS J. 2013; 280 (23167779): 285-30110.1111/febs.12069Crossref PubMed Scopus (60) Google Scholar, 20Biely P. Puchart V. Stringer M.A. Mørkeberg Krogh K.B.R Trichoderma reesei XYN VI: a novel appendage-dependent eukaryotic glucuronoxylan hydrolase.FEBS J. 2014; 281 (25041335): 3894-390310.1111/febs.12925Crossref PubMed Scopus (40) Google Scholar21Luo H. Yang J. Li J. Shi P. Huang H. Bai Y. Fan Y. Yao B. Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases.Appl. Microbiol. Biotechnol. 2010; 86 (20077114): 1829-183910.1007/s00253-009-2410-0Crossref PubMed Scopus (59) Google Scholar), as compared with bacterial GH30-8 enzymes, such as EcXynA (24.4%), BsXynC (23.3%), and CaXyn30A from Clostridium acetobutylicum (26.3%) (2St John F.J. Rice J.D. Preston J.F. Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and analysis of its role in depolymerization of glucuronoxylan.J. Bacteriol. 2006; 188 (17028274): 8617-862610.1128/JB.01283-06Crossref PubMed Scopus (99) Google Scholar, 12St John F.J. Dietrich D. Crooks C. Balogun P. de Serrano V. Pozharski E. Smith J.K. Bales E. Hurlbert J.C. A plasmid borne, functionally novel glycoside hydrolase family 30, subfamily 8 endoxylanase from solventogenic Clostridium.Biochem. J. 2018; 475 (29626157): 1533-155110.1042/BCJ20180050Crossref PubMed Scopus (18) Google Scholar, 22Hurlbert J.C. Preston 3rd, J.F. Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi.J. Bacteriol. 2001; 183 (11222610): 2093-210010.1128/JB.183.6.2093-2100.2001Crossref PubMed Scopus (71) Google Scholar). Two conserved catalytic residues previously identified in GH30 xylanase—a general acid/base residue and a nucleophilic residue—were found to correspond with Glu202 and Glu297, respectively, in Xyn30B (Fig. 1, gray highlights). As with XYN IV and XYN VI, an Arg residue responsible for recognition of the MeGlcA in GH30-8 enzymes is not conserved in Xyn30B (Fig. 1, red box). The Xyn30B amino acid sequence includes a signal sequence (residues 1–22) as predicted by the SignalP server (23Nielsen H. Predicting secretory proteins with signalP.Methods Mol. Biol. 2017; 1611 (28451972): 59-7310.1007/978-1-4939-7015-5_6Crossref PubMed Scopus (567) Google Scholar). The cleavage site of the signal peptide was estimated to lie between Ala19 and Ile20 or between Ala22 and Gln23. Eight of the N-glycosylation sites (Asn60, Asn88, Asn111, Asn154, Asn215, Asn334, Asn346, and Asn412) were predicted by the NetNglyc server (http://www.cbs.dtu.dk/services/NetNGlyc/). 3Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. Xyn30B was overexpressed using the T. cellulolyticus homologous expression system (24Inoue H. Fujii T. Yoshimi M. Taylor 2nd, L.E. Decker S.R. Kishishita S. Nakabayashi M. Ishikawa K. Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus.J. Ind. Microbiol. Biotechnol. 2013; 40 (23700177): 823-83010.1007/s10295-013-1286-2Crossref PubMed Scopus (34) Google Scholar). SDS-PAGE analysis of the purified enzyme showed a molecular mass slightly larger than 49,403 Da, which has been estimated from the primary structure excluding the N-terminal signal peptide (Fig. 2). Furthermore, the average molecular mass of Xyn30B determined by TOF-MS was 56,354 Da, indicating that Xyn30B was glycosylated at several sites. The glycosylation sites in Xyn30B were assigned by X-ray crystallography, as described below. Xyn30B exhibited xylanase activity on beechwood xylan (11.3 units mg−1) and birchwood xylan (9.0 units mg−1), whereas degradation activities for arabinoxylan, carboxymethyl cellulose, glucomannan, and xyloglucan were not detected by the 3,5-dinitrosalicylic acid (DNS) method. The optimum pH and temperature for hydrolysis of beechwood xylan were estimated around pH 4 and 50 °C, respectively (Fig. S2). Xyn30B retained more than 90% activity after incubation for 30 min at 40 °C in pH over a range of 3–6.5 and was stable for 24 h at temperatures below 40 °C at pH 4.0. The initial degradation product of beechwood xylan by Xyn30B was found to be acidic XOSs, whereas linear oligosaccharides and xylose were hardly detected (Fig. 3). These observations indicate that Xyn30B is a glucuronoxylan-specific xylanase. Xyn30B also degraded the MeGlcA-appended oligosaccharide analogue, borohydride-reduced aldotetrauronic acid (BR-MeGlcA3Xyl3), into aldotriuronic acid (MeGlcA2Xyl2) and xylitol. Kinetic parameters were also determined as follows: Km = 19 mg ml−1 and kcat = 17 s−1 for beechwood xylan; Km = 0.064 mm and kcat = 23 s−1 for BR-MeGlcA3Xyl3. The low Km value for BR-MeGlcA3Xyl3 suggests that Xyn30B has high affinity for MeGlcA. The molecular content in acidic XOS produced by Xyn30B was evaluated by ESI(−)-MS (Fig. 4A). Acidic products were readily observed as singly and doubly charged deprotonated species generically labeled [XylnMeGlcA − H]− (1−, mainly COO− from MeGlcA) and [XylnMeGlcA − 2H]2− (2−, COO− from MeGlcA and O− from anomeric carbon). They correspond to the XOS backbones formed by n xylose units and carrying one MeGlcA moiety with no information about its position. The ESI(−) analysis filters the oligoxylose species, Xyln, carrying no acidic moiety and allows for instant visualization of the shortest acidic product, which was found to be Xyl2MeGlcA at m/z 471 (Fig. 4A, highlighted in boldface red) and associated with a broad distribution of longer congeners up to Xyl14MeGlcA at m/z 1,027 (2−). The position of the MeGlcA moiety along the XOS chain (reducing end, nonreducing end, or in between) was further revealed using a multistage MS procedure (MSn). Upon activation in MS2 (Fig. 4B), the [Xyl2MeGlcA − H]− expelled a neutral C2H4O2 via a cross-ring cleavage, yielding 0,2A3 at m/z 411 (−60 Da) concomitantly to a xylose unit via a glycosidic bond cleavage that yielded a C2 at m/z 339 (−132 Da). As the precursor ion is two xylose units long, it instantly indicates that the MeGlcA is located at the nonreducing end of Xyl2 (Fig. 4C). Both cross-ring and glycosidic bond cleavages have been found to occur only at the reducing ends of deprotonated acidic products (25Reis A. Domingues M.R.M Domingues P. Ferrer-Correia A.J. Coimbra M.A. Positive and negative electrospray ionisation tandem mass spectrometry as a tool for structural characterisation of acid released oligosaccharides from olive pulp glucuronoxylans.Carbohydr. Res. 2003; 338 (12829395): 1497-150510.1016/S0008-6215(03)00196-4Crossref PubMed Scopus (39) Google Scholar, 26Fouquet T. Sato H. Nakamichi Y. Matsushika A. Inoue H. Electrospray multistage mass spectrometry in the negative ion mode for the unambiguous molecular and structural characterization of acidic hydrolysates from 4-O-methylglucuronoxylan generated by endoxylanases.J. Mass Spectrom. 2018; (30597672)10.1002/jms.432110.1002/jms.4321Google Scholar). The ESI(−)-MS3 spectrum of C2 displays two product ions formed upon its dehydration (B2 at m/z 321, −18 Da) and a cross-ring cleavage of the last xylose moiety (0,2X2 at m/z 249) as the two sole fragmentation pathways that are opened up by the MeGlcA position at the reducing end of the activated species (Fig. 4B). Both the cross-ring cleavage (yielding 0,2A4 at m/z 543) and the glycosidic bond cleavage (yielding C3 at m/z 471) were observed in the ESI(−)-MS2 fingerprint of the deprotonated acidic product, [Xyl3MeGlcA − H]−, at m/z 603 (Fig. 5A), indicating that the MeGlcA residue is not located at its reducing end. In the MS3 spectra (Fig. 5A), C3 was found to dissociate into B3 at m/z 453 (dehydration, −18 Da) and 0,2X2 only (cross-ring cleavage at the reducing end). Deviating from the MS2 spectrum of [Xyl2MeGlcA − H]− (Fig. 4B) but resembling the MS3 pattern of C2 from [Xyl2MeGlcA − H]−, it unambiguously localized the acidic MeGlcA pendant group at the reducing end of C3. In a reverse chain reconstruction, one xylose unit was added to the reducing end of C3, demonstrating that [Xyl3MeGlcA − H]− carries the glucuronic acid moiety one unit away from the reducing end (Fig. 5C). A similar conclusion was drawn for [Xyl5MeGlcA − H]− at m/z 867 (Fig. 5B); the ESI(−)-MS2 spectrum barely displayed a xylose-shorter C5 ion product at m/z 735 (glycosidic bond cleavage at the reducing end), indicating the MeGlcA is not located at the reducing end but probably one unit away, considering the low intensity of C5 (26Fouquet T. Sato H. Nakamichi Y. Matsushika A. Inoue H. Electrospray multistage mass spectrometry in the negative ion mode for the unambiguous molecular and structural characterization of acidic hydrolysates from 4-O-methylglucuronoxylan generated by endoxylanases.J. Mass Spectrom. 2018; (30597672)10.1002/jms.432110.1002/jms.4321Google Scholar). Its ESI(−)-MS3 fingerprint was eventually identical to the previous MS3 pattern of C3 from [Xyl3MeGlcA − H]− (Fig. 5A) with the B5 and 0,2X2 product ions only (Fig. 5B), suggesting that the MeGlcA is positioned at the reducing end of C5, one unit away for [Xyl5MeGlcA − H]−. This indicates a generic MeGlcA2Xyln (n > 1) shape for the all of the acidic products released using Xyn30B (Fig. 4A). From these results, Xyn30B appears to specifically cleave glucuronoxylan at the second glycosidic linkage from the MeGlcA residue toward the reducing end, similarly to typical GH30 glucuronoxylanases. During the hydrolysis of beechwood xylan by Xyn30B, we noticed that Xyl2 was produced after prolonged incubation and increased protein loading of the reaction mixture (Fig. 6A). The increases in MeGlcA2Xyl2 and MeGlcA2Xyl3 were also observed with a decrease in longer acidic XOS in the mixture. These results indicate that Xyl2 was produced by further degradation of the acidic XOS. The specific production of Xyl2 suggests that Xyn30B has xylobiohydrolase activity, releasing Xyl2 units from the acidic XOSs that were generated in the initial stage of the reaction. This activity was predicted to release the product from the nonreducing end of MeGlcA2Xyln. In addition, the production of xylotetraose (Xyl4) and xylohexaose (Xyl6) was significantly increased with increasing time compared with that of xylotriose (Xyl3) and xylopentaose (Xyl5) (Fig. 6A). The product concentrations are shown in Table S1. These observations imply that the xylobiohydrolase activity was accompanied by transglycosylation activity, which transfers the Xyl2 from the acidic XOS to the free acceptors (Xyl2 and Xyl4). The xylobiohydrolase activity of Xyn30B was also confirmed for linear XOS, which was MeGlcA appendage–independent. When Xyl3 was used as the substrate, xylose and Xyl2 were produced as hydrolysates, and Xyl5 was formed through a transglycosylation (Fig. 6B). The hydrolase and transglycosylation activities of Xyl3 were 0.388 and 0.303 units mg−1 for the production of xylose and Xyl5, respectively. The major products from Xyl4 were identified as Xyl2 and Xyl6. A small amount of unidentified XOS longer than Xyl6 was also observed during the hydrolysis of these substrates, probably due to further transglycosylation (Fig. 6B, arrows). In contrast, no products were produced when only Xyl2 was used as the substrate (data not shown), suggesting that transglycosylation occurs during the hydrolysis. These results indicate that Xyn30B is a bifunctional enzyme possessing both MeGlcA appendage–dependent glucuronoxylanase activity and xylobiohydrolase (including transglycosylation) activity. The crystal structure of Xyn30B was determined at 2.25 Å resolution by molecular replacement using CaXyn30A as the search model (PDB code 5CXP). The data collection and refinement statistics are shown in Table 1. The Xyn30B crystal was in the P212121 space group with two protein molecules (chains A and B) in the asymmetric unit. Amino acid residues numbered 20–473 were assigned to chains A and B with the electron density map indicating that the N-terminal signal sequence was cleaved between Ala19 and Ile20. Glu474, which is the C-terminal residue, could not be assigned due to disorder.Table 1Statistics for X-ray crystallographyData collectionWavelength (Å)0.9Resolution limits (Å)48.26–2.25 (2.28–2.25)aValues in parentheses are for the highest-resolution shell.Space groupP212121Unit cell dimensions a, b, c (Å)83.2, 114.9, 118.5No. of reflections370,211 (16,467)No. of unique reflections54,785 (2,649)Redundancy6.8 (6.2)Completeness (%)99" @default.
• W2910361751 created "2019-01-25" @default.
• W2910361751 creator A5006874069 @default.
• W2910361751 creator A5008092848 @default.
• W2910361751 creator A5018642962 @default.
• W2910361751 creator A5023003247 @default.
• W2910361751 creator A5060223039 @default.
• W2910361751 creator A5065988743 @default.
• W2910361751 date "2019-03-01" @default.
• W2910361751 modified "2023-09-30" @default.
• W2910361751 title "Structural and functional characterization of a bifunctional GH30-7 xylanase B from the filamentous fungus Talaromyces cellulolyticus" @default.
• W2910361751 cites W1491373961 @default.
• W2910361751 cites W1525274895 @default.
• W2910361751 cites W1601705790 @default.
• W2910361751 cites W1968766716 @default.
• W2910361751 cites W1968973081 @default.
• W2910361751 cites W1971323157 @default.
• W2910361751 cites W1979430872 @default.
• W2910361751 cites W1980780850 @default.
• W2910361751 cites W1988420174 @default.
• W2910361751 cites W1991329996 @default.
• W2910361751 cites W1997469639 @default.
• W2910361751 cites W2000163261 @default.
• W2910361751 cites W2008214748 @default.
• W2910361751 cites W2009674410 @default.
• W2910361751 cites W2012786918 @default.
• W2910361751 cites W2017911392 @default.
• W2910361751 cites W2029282790 @default.
• W2910361751 cites W2029629283 @default.
• W2910361751 cites W2032076607 @default.
• W2910361751 cites W2043123654 @default.
• W2910361751 cites W2056137000 @default.
• W2910361751 cites W2061242649 @default.
• W2910361751 cites W2065097897 @default.
• W2910361751 cites W2065905475 @default.
• W2910361751 cites W2071776850 @default.
• W2910361751 cites W2077055748 @default.
• W2910361751 cites W2088366138 @default.
• W2910361751 cites W2094646744 @default.
• W2910361751 cites W2096335011 @default.
• W2910361751 cites W2097493124 @default.
• W2910361751 cites W2103096659 @default.
• W2910361751 cites W2108921801 @default.
• W2910361751 cites W2120667106 @default.
• W2910361751 cites W2122559203 @default.
• W2910361751 cites W2124026197 @default.
• W2910361751 cites W2126967756 @default.
• W2910361751 cites W2127130852 @default.
• W2910361751 cites W2127322768 @default.
• W2910361751 cites W2140107840 @default.
• W2910361751 cites W2141920771 @default.
• W2910361751 cites W2149774761 @default.
• W2910361751 cites W2154762068 @default.
• W2910361751 cites W2165574256 @default.
• W2910361751 cites W2165973682 @default.
• W2910361751 cites W2180229411 @default.
• W2910361751 cites W2311203695 @default.
• W2910361751 cites W2599440902 @default.
• W2910361751 cites W2609207099 @default.
• W2910361751 cites W2758793736 @default.
• W2910361751 cites W2765322245 @default.
• W2910361751 cites W2791011553 @default.
• W2910361751 cites W2791179444 @default.
• W2910361751 cites W2795531422 @default.
• W2910361751 cites W4248872320 @default.
• W2910361751 doi "https://doi.org/10.1074/jbc.ra118.007207" @default.
• W2910361751 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6422087" @default.
• W2910361751 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30655295" @default.
• W2910361751 hasPublicationYear "2019" @default.
• W2910361751 type Work @default.
• W2910361751 sameAs 2910361751 @default.
• W2910361751 citedByCount "38" @default.
• W2910361751 countsByYear W29103617512019 @default.
• W2910361751 countsByYear W29103617512020 @default.
• W2910361751 countsByYear W29103617512021 @default.
• W2910361751 countsByYear W29103617512022 @default.
• W2910361751 countsByYear W29103617512023 @default.
• W2910361751 crossrefType "journal-article" @default.
• W2910361751 hasAuthorship W2910361751A5006874069 @default.
• W2910361751 hasAuthorship W2910361751A5008092848 @default.
• W2910361751 hasAuthorship W2910361751A5018642962 @default.
• W2910361751 hasAuthorship W2910361751A5023003247 @default.
• W2910361751 hasAuthorship W2910361751A5060223039 @default.
• W2910361751 hasAuthorship W2910361751A5065988743 @default.
• W2910361751 hasBestOaLocation W29103617511 @default.
• W2910361751 hasConcept C161790260 @default.
• W2910361751 hasConcept C171250308 @default.
• W2910361751 hasConcept C181199279 @default.
• W2910361751 hasConcept C185592680 @default.
• W2910361751 hasConcept C192562407 @default.
• W2910361751 hasConcept C2775859485 @default.
• W2910361751 hasConcept C2779678110 @default.
• W2910361751 hasConcept C2780443040 @default.
• W2910361751 hasConcept C2780841128 @default.
• W2910361751 hasConcept C2908559767 @default.
• W2910361751 hasConcept C31903555 @default.
• W2910361751 hasConcept C55493867 @default.
• W2910361751 hasConcept C59822182 @default.

• next