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 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 Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

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 Essential Role for the Phosphatidylinositol 3,5-Bisphosphate Synthesis Complex in Caspofungin Tolerance and Virulence in Candida glabrata

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Deepak Kumar Choudhary ◽  
Priyanka Bhakt ◽  
Rupinder Kaur
Keyword(s):  
Cell Wall ◽  
Candida Glabrata ◽  
Scaffolding Protein ◽  
Pathogenic Yeast ◽  
Lipid Kinase ◽  
Lipid Molecule ◽  
Content Type ◽  
Phosphoinositide Signaling ◽  
Promising Target ◽  
Opportunistic Fungal Pathogen

ABSTRACT Increasing resistance of the human opportunistic fungal pathogen Candida glabrata toward the echinocandin antifungals, which target the cell wall, is a matter of grave clinical concern. Echinocandin resistance in C. glabrata has primarily been associated with mutations in the β-glucan synthase-encoding genes C. glabrata FKS1 (CgFKS1) and CgFKS2. This notwithstanding, the role of the phosphoinositide signaling in antifungal resistance is just beginning to be deciphered. The phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance lipid molecule that is pivotal to the intracellular membrane traffic. Here, we demonstrate for the first time that the PI(3,5)P2 kinase CgFab1, along with its activity regulator CgVac7 and the scaffolding protein CgVac14, is required for maintenance of the cell wall chitin content, survival of the cell wall, and caspofungin stress. Further, deletion analyses implicated the PI(3,5)P2 phosphatase CgFig4 in the regulation of PI(3,5)P2 levels and azole and echinocandin tolerance through CgVac14. We also show the localization of the CgFab1 lipid kinase to the vacuole to be independent of the CgVac7, CgVac14, and CgFig4 proteins. Lastly, our data demonstrate an essential requirement for PI(3,5)P2 signaling components, CgFab1, CgVac7, and CgVac14, in the intracellular survival and virulence in C. glabrata. Altogether, our data have yielded key insights into the functions and metabolism of PI(3,5)P2 lipid in the pathogenic yeast C. glabrata. In addition, our data highlight that CgVac7, whose homologs are absent in higher eukaryotes, may represent a promising target for antifungal therapy.

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 Characterization of Two Distinct Glycosyl Hydrolase Family 78 α-l-Rhamnosidases from Pediococcus acidilactici

2011 ◽  
Vol 77 (18) ◽  
pp. 6524-6530 ◽  
Author(s):  
Herbert Michlmayr ◽  
Walter Brandes ◽  
Reinhard Eder ◽  
Christina Schümann ◽  
Andrés M. del Hierro ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Pediococcus Acidilactici ◽  
Glycosyl Hydrolase Family ◽  
Negative Effects ◽  
Content Type ◽  
Linalool Oxide ◽  
Hydrolysis Of ◽  
Flavanone Glycoside ◽  
Hydrolase Family

ABSTRACTα-l-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ramandram2) fromPediococcus acidilacticiDSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial β-glucosidase, both enzymes released the monoterpenes linalool andcis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substratep-nitrophenyl-α-l-rhamnopyranoside (Vmax= 243 U mg−1). In contrast, Ram2 displayed considerable specificity toward hesperidin (Vmax= 34 U mg−1) and, especially, rutinose (Vmax= 1,200 U mg−1), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases.

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