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PubMed2004

The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides.

The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall. A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction. The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome. The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome. The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction. In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex.

Amino Acid Sequence↗
PubMed1998

Cellulosomes-structure and ultrastructure.

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module-the cellulose-binding domain-is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelope via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dynamic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are then taken up and assimilated by normal cellular processes.

Cell Membrane↗
PubMed2003

Two new cellulosome components encoded downstream of celI in the genome of Clostridium thermocellum: the non-processive endoglucanase CelN and the possibly structural protein CseP.

Clostridium thermocellum produces a great number of extracellular cellulases which are free or cellulosome-bound. The nucleotide sequence of a gene cluster containing the genes celI, celN and cseP was determined from C. thermocellum strain F7. Gene products Cel9I and Cel9N are structurally related enzymes having a glycosyl hydrolase family 9 and a carbohydrate-binding module (CBM3c), but show characteristic differences: Cel9I is a non-cellulosomal protein with an additional CBM (CBM3b), whereas Cel9N contains a cellulosomal dockerin module and no additional CBM. Although Cel9I is a processive endoglucanase, Cel9N is non-processive. Both enzymes hydrolyse phosphoric acid swollen cellulose, but the products of hydrolysis are different. The CseP protein encoded in the gene cluster is the first component attached to the cellulosomal scaffoldin for which no catalytic activity could be detected. It was shown to be present in the cellulosome. Its sequence is homologous to the spore-coat assembly protein CotH of Bacillus subtilis, suggesting a structural role of CseP in the cellulosome.

Bacterial Proteins↗
PubMed2004

Architecture of the Bacteroides cellulosolvens cellulosome: description of a cell surface-anchoring scaffoldin and a family 48 cellulase.

A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.

5' Flanking Region↗
PubMed2003

Structural organization of the intact bacterial cellulosome as revealed by electron microscopy.

The architecture of the intact cellulosome of Clostridium thermocellum, a huge extracellular multi-polypetide bacterial enzyme complex engaged in degradation of cellulose, was investigated by electron microscopy. This was done because former electron microscopic studies aimed at elucidation of the structure of polycellulosomes and cellulosomes were restricted by the fact that data on macromolecular details could only be derived from deformed or disrupted enzyme complexes, or by application of cryo preparation and imaging techniques yielding insufficient resolution. The shape of well-preserved cellulosomes was more or less spherical, often similar to that of an olive fruit with a cavity. Therein, multiple fibrillar structures could be visualized, interpreted to be the proximal stretches of copies of the fibrillar protein Cip A ('scaffoldin'), the nonenzymatic scaffolding protein known to function as attachment site for the enzymatic subunits, as well as fibrillar parts of anchoring proteins. The enzymatic subunits were depicted to be attached, in a repetitive fashion, to the distal stretches of the Cip A proteins. The enzymatic subunits were seen, in the intact cellulosome, to form a shell-like complex substructure surrounding the cavity. Obviously, this kind of architecture makes sure that the catalytic domains of the enzymatic subunits are exposed to the environment, and, hence, to the substrate, the cellulose fibrils. Attempts were made to demonstrate the alternating occurrence of coiled domains and fibrillar stretches along the elongated protein Cip A previously characterized by sequencing, X-ray, and NMR studies. To this end, Cip A molecules, with adhering enzymatic subunits, were partially removed from their native location within the cellulosome, "stretched" by hydromechanical forces directly on the electron microscopic support film, negatively stained, and depicted by electron microscopy. The alternating occurrence of presumed coiled domains and fibrillar stretches along Cip A could be visualized, together with detached enzymatic subunits found on the support film.

Bacterial Physiological Phenomena↗
PubMed2006

The functional repertoire of prokaryote cellulosomes includes the serpin superfamily of serine proteinase inhibitors.

Many of the Firmicutes bacteria responsible for plant polysaccharide degradation in Nature produce a multiprotein complex called a cellulosome, which co-ordinates glycoside hydrolase assembly, bacterial adhesion to substrate and polysaccharide hydrolysis. Cellulosomal proteins possess a dockerin module, which mediates their attachment to the scaffoldin protein via its interaction with cohesin modules, and only glycoside hydrolases and other carbohydrate active enzymes were known to reside within the cellulosome. We show here with Clostridium thermocellum ATCC 27405 that members of the serpin superfamily of serine proteinase inhibitors, which are best recognized for their conformational flexibility and co-ordination of key regulatory functions in multicellular eukaryotes, also reside within the cellulosome. These studies are the first to expand the cellulosome paradigm of protein complex assembly beyond glycoside hydrolase and carbohydrate active enzymes, and to include a newly identified functionality in the Firmicutes.

Amino Acid Sequence↗
PubMed2005

Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes.

Clostridium thermocellum produces the most efficient enzyme-complex for the degradation of polysaccharides in biomass, the large extracellular cellulosome. The draft complete genomic sequence of Clostridium thermocellum was screened for open reading frames (ORF) containing cellulosomal dockerin sequences. Seventy-one putative cellulosomal genes were detected. One third of these ORFs may be involved in cellulose hydrolysis. Most of the others showed homology to hemicellulases, pectinases, chitinases, glycosidases or esterases potentially involved in the unwrapping of cellulose fibers. To identify the predominant catalytic components, cellulosomes were purified and the components were separated by an adapted two-dimensional gel electrophoresis technique. The apparent major spots were identified by MALDI-TOF/TOF. Ten of the components were previously known: the structural protein CipA, the endo-glucanases Cel8A, Cel5G, Cel9N, the cellobiohydrolases Cbh9A, Cel9K, Cel48S, the xylanases Xyn10C, Xyn10Z, and the chitinase Chi18A. In addition, three hitherto unknown major components were detected, Cel9R, Xyn10D and Xgh74A. These major components in the cellulosomal particles most probably constitute the essential enzymes for crystalline cellulose hydrolysis.

Catalysis↗
PubMed1997

Determination of immunological homology between cellulosome subunits and cloned endoglucanases and xylanase of Clostridium thermocellum.

Immuno-cross reactivity between the subunits of Clostridium thermocellum cellulosome and cloned endogucanases and xylanase from the same bacterium was studied using the polyclonal antibodies raised against cloned enzymes. Dot blot analysis showed that the cellulosome, S8 and S11 subunits cross-reacted strongly with the antibodies of all cloned enzymes tested except that raised against CelC. Western blot analysis revealed that S8 and S11 subunits cross-reacted with the antibodies of CelA, CelB, CelD, CelG, CelH and XynZ, but the antibodies of CelB and CelG were highly specific for S8 and S11 subunits, respectively. Similar analysis using dissociated cellulosome showed that the antibodies of all cloned enzymes tested cross-reacted with more than one subunit of the cellulosome. Antibodies of CelC showed a very low cross-reactivity against all subunits of the cellulosome. The results indicate that immunological cross-reactivity studies could be useful, not only for demonstrating the similarities between native and cloned enzymes, but also for identifying native enzymes using antibodies of cloned enzymes.

Journal Article↗
PubMed1991

Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components.

The function of the non-catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross-reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I-labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I-labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.

Animals↗
PubMed1996

Structural role of calcium for the organization of the cellulosome of Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is a multipolypeptide complex of structural and catalytic subunits. Several of the catalytic subunits have at the carboxyl end a conserved duplicated region (CDR) which interacts with internally repeated elements (IREs) of scaffolding subunits such as CipA. This interaction requires calcium. The two parts of the CDR region here designated CDR1 and CDR2 (closest to the carboxyl end) each consist of about 20 amino acids residues. As shown in our previous paper [Choi, S.K., & Ljungdahl, L.G. (1996) Biochemistry 35, 4897-4905], treatment of the cellulosome with ethylenediaminetetraacetic acid (EDTA) under aerobic conditions disintegrates the cellulosome with formation of truncated catalytic subunits. The cleavage is at a specific asparagine residue located within CDR1 and occurs with complete loss of CDR2. Two branched peptides containing the amino acid sequences of CDR1 and CDR2 (designated bCDR1 and bCDR2) were synthesized, and specific antibodies were raised against them. These antibodies did not cross react with bCDR1 or bCDR2, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, it was observed that about 15 subunits of the cellulosome reacted with anti-bCDR1 and anti-bCDR2. In a similar experiment with EDTA-treated cellulosomes, these subunits reacted with anti-bCDR1 but not with anti-bCDR2, showing that they lost the bCDR2 epitope and were truncated. The peptide bCDR1 binds calcium, whereas bCDR2 does not. Furthermore, bCDR1 but not bCDR2 binds to CipA, presumably at IRE regions. This binding requires calcium. A model is proposed for the binding of the catalytic subunits to CipA which involves CDR1, an IRE, and calcium.

Amino Acid Sequence↗
PubMed2002

An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2.

The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components. To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

Amino Acid Sequence↗
PubMed2004

Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum.

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.

Base Sequence↗
PubMed2002

Determination of subunit composition of Clostridium cellulovorans cellulosomes that degrade plant cell walls.

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.

Amino Acid Sequence↗
PubMed1985

Organization and distribution of the cellulosome in Clostridium thermocellum.

The properties of the cellulosome (the cellulose-binding, multicellulase-containing protein complex) in Clostridium thermocellum were examined by comparing the cellulase systems derived from the wild type and an adherence-defective mutant. The growth conditions--specifically, growth either on cellulose (Avicel) or on cellobiose as insoluble or soluble carbon sources, respectively--were found to be critical to the distribution of the cellulosome in the mutant system: the cellobiose-grown mutant (in contrast to the wild type) lacked the cellulosome on its surface and produced only minor quantities of the extracellular cellulosome accompanied by other relatively low-molecular-weight cellulases. The polypeptide composition of the respective purified cellulosome was dependent on the nature of the carbon source and was similar for both wild-type and mutant cells. Ultrastructural analysis revealed the presence of novel polycellulosomal protuberances on the cell surface of the cellobiose-grown wild type which were absent in the mutant.

Cell Membrane↗
PubMed1986

Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose.

The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy. When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures. However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate). In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate. The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.

Bacterial Proteins↗
PubMed1990

Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes.

Xylanase activity of Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, was characterized. The activity was localized both in the cellulosome (the principal multienzyme, cellulose-solubilizing protein complex) and in noncellulosomal fractions. Each of these fractions contained at least four major polypeptide bands which contributed to the xylanolytic activity. In both cases, pH and temperature optima, product pattern, and other features of the xylanase activity were almost identical. The main difference was in the average molecular weights of the respective polypeptides which appeared responsible for the activity. In the noncellulosomal fraction, xylanases with Mrs ranging from 30,000 to 65,000 were detected. Distinct from these were the cellulosomal xylanases, which exhibited much larger Mrs (up to 170,000). The cellulosome-associated xylanases corresponded to known cellulosomal subunits, some of which also exhibited endoglucanase activity, and others which coincided with subunits which appeared to express exoglucanaselike activity. In contrast, the noncellulosomal xylanases hydrolyzed xylan exclusively. beta-Glucosidase and beta-xylosidase activities were shown to be the action of different enzymes; both were associated exclusively with the cell and were not components of the cellulosome. Despite the lack of growth on and utilization of xylan or its degradation products, C. thermocellum produces a highly developed xylanolytic apparatus which is interlinked with its cellulase system.

Bacterial Adhesion↗
PubMed1991

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.

Animals↗
PubMed1999

The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production.

In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.

Bacterial Adhesion↗