Measurements of single molecular affinity interactions between carbohydrate-binding modules and crystalline cellulose fibrils

2013 ◽  
Vol 15 (17) ◽  
pp. 6508 ◽  
Author(s):  
Mengmeng Zhang ◽  
Bin Wang ◽  
Bingqian Xu
Keyword(s):  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Cellulose Fibrils ◽  
Molecular Affinity

Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan

2007 ◽  
Vol 8 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Lada Filonova ◽  
Åsa M. Kallas ◽  
Lionel Greffe ◽  
Gunnar Johansson ◽  
Tuula T. Teeri ◽  
...  
Keyword(s):  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Pulp Fibers ◽  
Carbohydrate Binding Modules

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.

scholarly journals Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules

2019 ◽  
Vol 116 (45) ◽  
pp. 22545-22551 ◽  
Author(s):  
Vera Novy ◽  
Kevin Aïssa ◽  
Fredrik Nielsen ◽  
Suzana K. Straus ◽  
Peter Ciesielski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Degradation Mechanism ◽  
Length Distribution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Distribution Analysis ◽  
Cellulose Accessibility ◽  
Carbohydrate Binding Modules

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

scholarly journals Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Jordan F. Russell ◽  
Matthew L. Russo ◽  
Xuewen Wang ◽  
Neal Hengge ◽  
Daehwan Chung ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Plant Biomass ◽  
Protein Glycosylation ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Unique Gene ◽  
Glycosyl Transferase ◽  
Caldicellulosiruptor Bescii ◽  
Carbohydrate Binding Modules

ABSTRACT Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases. IMPORTANCE Caldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

scholarly journals In situ detection of cell wall polysaccharides in sitka spruce (Picea sitchensis (Bong.) Carrière) wood tissue

BioResources ◽  
2007 ◽  
Vol 2 (2) ◽  
pp. 284-295
Author(s):  
Clemens Altaner ◽  
J. Paul Knox ◽  
Michael C. Jarvis
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Cell Walls ◽  
Sitka Spruce ◽  
Picea Sitchensis ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Cell Wall Polysaccharides ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules

Wood cell wall polysaccharides can be probed with monoclonal antibodies and carbohydrate-binding modules (CBMs). Binding of monoclonal antibodies to β-1-4-xylan, β-1-4-mannan, β-1-3-glucan, and α-1-5-arabinan structures were observed in native Sitka spruce (Picea sitchensis (Bong.) Carrière) wood cell walls. Furthermore CBMs of different families, differing in their affinities for crystalline cellulose (3a) and amorphous cellulose (17 and 28), were shown to bind to the native wood cell walls with varying intensities. Resin channel forming cells exhibited an increased β-1-4-xylan and a decreased β-1-4-mannan content. Focusing on severe compression wood (CW) tracheids, β-1-3-glucan was found towards the cell lumen. In contrast, α-1-5-arabinan structures were present in the intercellular spaces between the round tracheids in severe CW, highlighting the importance of this polymer in cell adhesion.

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.

Labeling the planar face of crystalline cellulose using quantum dots directed by type-I carbohydrate-binding modules

Cellulose ◽  
2008 ◽  
Vol 16 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Qi Xu ◽  
Melvin P. Tucker ◽  
Phil Arenkiel ◽  
Xin Ai ◽  
Garry Rumbles ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Type I ◽  
Carbohydrate Binding Modules

scholarly journals Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

1999 ◽  
Vol 345 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Maher ABOU HACHEM ◽  
Eva NORDBERG KARLSSON ◽  
Eva BARTONEK-ROXÅ ◽  
Srinivasrao RAGHOTHAMA ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Binding Affinities ◽  
Binding Assays ◽  
Rhodothermus Marinus ◽  
Binding Studies ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules ◽  
Cellulose Binding

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichiacoli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module

2001 ◽  
Vol 355 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Ian E. BROWN ◽  
Marie H. MALLEN ◽  
Simon J. CHARNOCK ◽  
Gideon J. DAVIES ◽  
Gary W. BLACK
Keyword(s):  
Sequence Similarity ◽  
Functional Module ◽  
Pectate Lyase ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
High Sequence Identity ◽  
Pectate Lyases ◽  
Carbohydrate Binding Modules ◽  
The Family ◽  
Catalytic Module

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH10 and in the presence of 2mM Ca2+. Highest enzyme activity was detected at 62°C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167–177], are the first CBM-containing pectinases described to date.

Structure of a family 3b′ carbohydrate-binding module from the Cel9V glycoside hydrolase fromClostridium thermocellum: structural diversity and implications for carbohydrate binding

2009 ◽  
Vol 66 (1) ◽  
pp. 33-43 ◽  
Author(s):  
Svetlana Petkun ◽  
Sadanari Jindou ◽  
Linda J. W. Shimon ◽  
Sonia Rosenheck ◽  
Edward A. Bayer ◽  
...  
Keyword(s):  
Structural Diversity ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Asymmetric Unit ◽  
Space Group ◽  
Carbohydrate Binding Modules ◽  
Planar Array ◽  
Binding Residues ◽  
Cellulose Binding

Family 3 carbohydrate-binding modules (CBM3s) are associated with both cellulosomal scaffoldins and family 9 glycoside hydrolases (GH9s), which are multi-modular enzymes that act on cellulosic substrates. CBM3s bind cellulose. X-ray crystal structures of these modules have established an accepted cellulose-binding mechanism based on stacking interactions between the sugar rings of cellulose and a planar array of aromatic residues located on the CBM3 surface. These planar-strip residues are generally highly conserved, although some CBM3 sequences lack one or more of these residues. In particular, CBM3b′ fromClostridium thermocellumCel9V exhibits such sequence changes and fails to bind cellulosic substrates. A crystallographic investigation of CBM3b′ has been initiated in order to understand the structural reason(s) for this inability. CBM3b′ crystallized in space groupC2221(diffraction was obtained to 2.0 Å resolution in-house) with three independent molecules in the asymmetric unit and in space groupP41212 (diffraction was obtained to 1.79 Å resolution in-house and to 1.30 Å resolution at a synchrotron) with one molecule in the asymmetric unit. The molecular structure of Cel9V CBM3b′ revealed that in addition to the loss of several cellulose-binding residues in the planar strip, changes in the backbone create a surface `hump' which could interfere with the formation of cellulose–protein surface interactions and thus prevent binding to crystalline cellulose.

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