scholarly journals Domain Analysis of a Modular α-l-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85

2010 ◽  
Vol 192 (20) ◽  
pp. 5424-5436 ◽  
Author(s):  
Shosuke Yoshida ◽  
Charles W. Hespen ◽  
Robert L. Beverly ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Plant Cell Wall ◽  
Catalytic Efficiency ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Biofuel Production ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Carbohydrate Binding Module ◽  
Degrading Bacterium ◽  
Catalytic Module

ABSTRACT Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k cat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the Km for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

scholarly journals A mannanase, ManA, of the polycentric anaerobic fungusOrpinomycessp. strain PC-2 has carbohydrate binding and docking modules

2005 ◽  
Vol 51 (7) ◽  
pp. 559-568 ◽  
Author(s):  
Eduardo A Ximenes ◽  
Huizhong Chen ◽  
Irina A Kataeva ◽  
Michael A Cotta ◽  
Carlos R Felix ◽  
...  
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Anaerobic Fungus ◽  
Ph Profile ◽  
Catalytic Module ◽  
Cellulose Binding

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or β-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 °C, but was rapidly inactivated at 60 °C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.Key words: Orpinomyces, anaerobic fungi, mannanase, cellulose-binding module, cellulosome.

scholarly journals Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

2016 ◽  
Vol 113 (26) ◽  
pp. 7136-7141 ◽  
Author(s):  
Immacolata Venditto ◽  
Ana S. Luis ◽  
Maja Rydahl ◽  
Julia Schückel ◽  
Vânia O. Fernandes ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Plant Cell Wall ◽  
Industrial Process ◽  
Site Directed Mutagenesis ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Ruminococcus Flavefaciens ◽  
Pectic Polysaccharide ◽  
Degrading Bacterium ◽  
Carbohydrate Binding Modules

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

scholarly journals Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

2009 ◽  
Vol 192 (2) ◽  
pp. 483-493 ◽  
Author(s):  
Shosuke Yoshida ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Unknown Function ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Rumen Bacterium ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Carbohydrate Binding Module ◽  
Active Site Residues ◽  
Acetyl Xylan Esterase ◽  
Binding Studies

ABSTRACT Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser44, His273, Glu194, and Asp270, with both Glu194 and Asp270 functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.

Effect of Family 22 Carbohydrate-Binding Module on the Thermostability of Xyn10B Catalytic Module fromClostridium stercorarium

2006 ◽  
Vol 70 (12) ◽  
pp. 3039-3041 ◽  
Author(s):  
Rie ARAKI ◽  
Shuichi KARITA ◽  
Akiyoshi TANAKA ◽  
Tetsuya KIMURA ◽  
Kazuo SAKKA
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Catalytic Module

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

2013 ◽  
Vol 168 (4) ◽  
pp. 403-408 ◽  
Author(s):  
Muhammad Imran M. Khan ◽  
Muhammad Sajjad ◽  
Saima Sadaf ◽  
Rehan Zafar ◽  
Umer H.K. Niazi ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Catalytic Efficiency ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

scholarly journals NMR elucidation of nonproductive binding sites of lignin models with carbohydrate-binding module of cellobiohydrolase I

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yuki Tokunaga ◽  
Takashi Nagata ◽  
Keiko Kondo ◽  
Masato Katahira ◽  
Takashi Watanabe
Keyword(s):  
Binding Sites ◽  
Specific Interaction ◽  
Enzymatic Saccharification ◽  
Cost Effective ◽  
Biofuel Production ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Cellobiohydrolase I ◽  
Methoxy Groups ◽  
Nonproductive Binding

Abstract Background Highly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated. Results Three types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H–13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π–π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16–4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. Conclusion The CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

scholarly journals Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

2012 ◽  
Vol 78 (11) ◽  
pp. 3923-3931 ◽  
Author(s):  
Susana Valeria Valenzuela ◽  
Pilar Diaz ◽  
F. I. Javier Pastor
Keyword(s):  
Catalytic Activity ◽  
Kinetic Parameters ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Dimensional Structure ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Catalytic Module

ABSTRACTXyn30D from the xylanolytic strainPaenibacillus barcinonensishas been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing aKmof 14.72 mg/ml and akcatvalue of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

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