scholarly journals Hydrophilic Domains of Scaffolding Protein CbpA Promote Glycosyl Hydrolase Activity and Localization of Cellulosomes to the Cell Surface of Clostridium cellulovorans

2004 ◽  
Vol 186 (19) ◽  
pp. 6351-6359 ◽  
Author(s):  
Akihiko Kosugi ◽  
Yoshihiko Amano ◽  
Koichiro Murashima ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Cell Walls ◽  
Cellulose Degradation ◽  
Glycosyl Hydrolase ◽  
Scaffolding Protein ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Primary Role ◽  
Clostridium Cellulovorans

ABSTRACT CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.

scholarly journals Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

2002 ◽  
Vol 184 (4) ◽  
pp. 884-888 ◽  
Author(s):  
Akihiko Kosugi ◽  
Koichiro Murashima ◽  
Yutaka Tamaru ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Surface Layer ◽  
Cell Surface ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Sodium Dodecyl ◽  
Scaffolding Protein ◽  
Clostridium Cellulovorans ◽  
Surface Anchoring

ABSTRACT engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.

scholarly journals Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans

2002 ◽  
Vol 184 (18) ◽  
pp. 5088-5095 ◽  
Author(s):  
Koichiro Murashima ◽  
Akihiko Kosugi ◽  
Roy H. Doi
Keyword(s):  
Synergistic Effect ◽  
Cellulose Degradation ◽  
Synergistic Effects ◽  
Glycosyl Hydrolase ◽  
The Other ◽  
Scaffolding Protein ◽  
Crystalline Cellulose ◽  
Initial Reaction ◽  
Clostridium Cellulovorans ◽  
Degradation Reactions

ABSTRACT Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.

scholarly journals Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans

2013 ◽  
Vol 79 (19) ◽  
pp. 5942-5948 ◽  
Author(s):  
Sang Duck Jeon ◽  
Ji Eun Lee ◽  
Su Jung Kim ◽  
Sung Hyun Park ◽  
Gi-Wook Choi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Glycosyl Hydrolase ◽  
Surface Display ◽  
Scaffolding Protein ◽  
Crystalline Cellulose ◽  
Unique Contribution ◽  
Surface Plasmon Resonance Analysis ◽  
Clostridium Cellulovorans ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACTThe cellulosomes produced byClostridium cellulovoransare organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain inC. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces ofC. cellulovoransusing its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that inC. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

scholarly journals Synergistic Interaction of Clostridium cellulovorans Cellulosomal Cellulases and HbpA

2007 ◽  
Vol 189 (20) ◽  
pp. 7190-7194 ◽  
Author(s):  
Satoshi Matsuoka ◽  
Hideaki Yukawa ◽  
Masayuki Inui ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Surface Layer ◽  
Cell Surface ◽  
Anaerobic Bacterium ◽  
Synergistic Interaction ◽  
Corn Fiber ◽  
Scaffolding Protein ◽  
Type I ◽  
Clostridium Cellulovorans ◽  
Insoluble Polysaccharides

ABSTRACT Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.

scholarly journals Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls

2002 ◽  
Vol 68 (4) ◽  
pp. 1610-1615 ◽  
Author(s):  
Koichiro Murashima ◽  
Akihiko Kosugi ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Scaffolding Protein ◽  
Plant Cell Walls ◽  
Higher Plant ◽  
Clostridium Cellulovorans ◽  
Plant Cell Wall Degradation

ABSTRACT Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.

scholarly journals Synergistic Effects of Cellulosomal Xylanase and Cellulases from Clostridium cellulovorans on Plant Cell Wall Degradation

2003 ◽  
Vol 185 (5) ◽  
pp. 1518-1524 ◽  
Author(s):  
Koichiro Murashima ◽  
Akihiko Kosugi ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Synergistic Effects ◽  
Complex Structure ◽  
Molar Ratio ◽  
Crystalline Cellulose ◽  
Cell Wall Degradation ◽  
Clostridium Cellulovorans

ABSTRACT Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from Clostridium cellulovorans. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation. The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases. Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions. The possible mechanism of synergism between XynA and cellulases is discussed.

scholarly journals Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Narumon Tangthirasunun ◽  
David Navarro ◽  
Sona Garajova ◽  
Didier Chevret ◽  
Laetitia Chan Ho Tong ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Podospora Anserina ◽  
Cellulosic Biomass ◽  
Striking Difference ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Wild Type ◽  
Content Type

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

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 New insights into Dutch Elm Disease: cell wall compositional, ecophysiological, vascular and nanomechanical assessments

2019 ◽  
Vol 25 (1) ◽  
pp. 10-14
Author(s):  
Jaroslav Durkovic ◽  
František Kačik ◽  
Miroslava Mamonova ◽  
Rastislav Lagana ◽  
Iigrid Canova ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cellulose Degradation ◽  
Leaf Gas Exchange ◽  
Leaf Growth ◽  
Dutch Elm Disease ◽  
Crystalline Cellulose ◽  
Flow Density ◽  
Tracheary Elements ◽  
Leaf Trait ◽  
Leaf Midrib

Comprehensive assessments were made of the chemical profiles of woody cell wall components, and also leaf growth, ecophysiological, vascular and nanomechanical traits for two Dutch elm hybrids 'Groeneveld' and 'Dodoens' which possess contrasting tolerances toward Dutch elm disease. Upon infection with Ophiostoma novo-ulmi ssp. americana × novo-ulmi, medium-molecular weight macromolecules of cellulose were degraded in both hybrids. A loss of crystalline and non-crystalline cellulose regions occurred in parallel. In 'Groeneveld' plants, syringyl-rich lignin provided a far greater degree of protection from cellulose degradation, but only guaiacyl-rich lignin in 'Dodoens' plants was involved in a successful defence against the fungus. Unexpectedly, we found a very high proportion of non-significant differences between the infected and non-infected plants of 'Dodoens', including similarities in leaf growth, leaf gas exchange and leaf midrib vascular traits, as well as in the nanomechanical properties of the cell walls of tracheary elements such as modulus of elasticity, adhesion and energy dissipation. Three years after initial inoculations, except for a few traits such as leaf slenderness, relative chlorophyll content, transpiration rate and sap flow density in branches, we found no evidence of a decrease in leaf trait performances among the infected plants of 'Dodoens', despite the occasional persistence of fungal hyphae in the lumens of leaf midrib tracheary elements.

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