Anaerobic bacteria secrete a large range of plant cell wall hydrolases, which are organised in multi-enzyme complexes termed cellulosomes.

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| Due to the efficiency of cellulosomes in degrading the plant cell wall there’s been an extensive of effort to understand how these mega-Dalton nanomachines work and how they could be used to obtain valuable products from low-cost biomass or agricultural waste.

 

On its basics, cellulosomes are composed by five different components:

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• The scaffoldin subunit: The scaffoldin subunit is a non-catalytic protein that contains one or more cohesin modules connected to other types of functional modules. Depending on the scaffoldin protein, the referred modules include a cellulose-specific carbohydrate-binding module, a dockerin, an X module of unknown function, an S-layer homology (SLH) module or a sortase anchoring motif. The scaffoldin is responsible for organising the different subunits into the complex, therefore, shaping the overall architecture of the cellulosome. Motional freedom of the scaffoldin subunit allows precise positioning of the catalytic modules according to the topography of the substrate.

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• The cohesin modules: Cohesin modules are the major building blocks of the scaffoldin subunit and are responsible for organising the cellulolytic subunits into the multi-enzyme complex. Cohesins are classified into three groups: type I, type II and type III, according to their phylogenetic similarity. type I cohesins are located in the scaffoldin subunit and are responsible for incorporating the different catalytic subunits; type II cohesins are located at the cell surface and are responsible for anchoring the multi-enzyme complex into the cell wall; type III cohesins still have an unclear function.

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• The dockerin modules: Dockerins are non-catalytic proteins with approximately 70 amino acids that contain two duplicated segments of about 22 residues and display internal two-fold symmetry, consisting of a duplicated F-hand calcium-binding motif. Dockerins specifically bind to determined type of cohesin and, therefore, they are named after them. As a result we have type I, II and III dockerins that bind to type I, II and III cohesins, respectively. Essentially, the dockerin modules act as anchors: they anchor the catalytic subunits to the scaffoldin protein (type I) and anchor the scaffoldin protein to the cell wall (type II). The function of type III dockerins is still unknown. Although structurally related, type I cohesins and dockerins were shown to be different from type II and do not cross react.

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• The catalytic modules: Cellulosomes contain an amazing diversity of enzymes that is proportional to the complexity of plant cell wall. In this sense, the array of polysaccharides presented by the plant cell walls is matched by the complexity and diversity of the cellulosomal catalytic machinery. The catalytic modules include glycoside hydrolases (GHs), glycosyltransferases (GTs), carbohydrate esterases (CE) and polysaccharide lyases (PL).

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• The carbohydrate-biding modules: Carbohydrate-binding modules (CBMs) are non-catalytic proteins that bind to a wide range of poly- and oligosaccharides. Their main function is to increase the activity of the associated catalytic modules by maintaining the enzyme in the proximity of the substrate through their sugar-binding activity. Furthermore, they are also responsible for anchoring the cellulosome to the substrate (targeting function) and for breaking the substrate (disruptive function).

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The cohesin-dockerin interaction

The cellulosome architecture is defined by high affinity protein-protein interactions between cohesins and dockerins. Dockerin and cohesin domains have been identified as conserved homologous sequence elements of the proteins that make up the cellulosome scaffold and enzymatic subunits.

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Dockerins are non-catalytic proteins of approximately 60-70 amino acids that recognise cohesin domains and mediate the assembly of the cellulolytic subunits into the scaffoldin subunit and of the latter to the bacterial cell wall. The dockerin sequence is highly conserved and made up of two 22-residue sequence repeats separated by a linker region of about 9-18 residues. They fold into three α-helices, with helices 1 and 3 comprising the repeated segments. Within each duplicated sequence there is a 12-residue segment with sequence similarity to the calcium-binding loop of the EF-hand motif, in which all the calcium binding residues are highly conserved.

The residues that coordinate calcium (aspartate or asparagine) are conserved in loop positions 1, 3, 5, 9, and 12 of nearly all dockerins. The presence of the duplicated segment suggests that both halves of the dockerin are able to interact with the cohesin in very a similar manner. This means that there may be plasticity in cohesin recognition by the dockerin with either the N- or C-terminal helix.  This plasticity allows, in principle, the simultaneous binding of two cohesins by a single dockerin. Such an interaction would not only provide a higher level of structure to the cellulosome but might also allow the crosslinking of two scaffoldins through a single dockerin. Nevertheless, the stoichiometry of type I cohesin-dockerin binding is, invariably 1:1, suggesting that the two binding sites are not able to bind simultaneously. Thus, it remains unclear the biological significance of the dual binding mode in dockerins. 

 

| "...the duplicated segment suggests that both halves of the dockerin are able to interact with the cohesin..."

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Cohesins are 150-residue modules, usually present as tandem repeats in scaffoldins. They are elongated, conical molecules that comprise a jelly-roll topology that folds into a nine-stranded β-sandwich. The cohesin modules are the main components of the scaffoldin subunit and are responsible for organising the cellulolytic subunits into the cellulosome. According to their phylogenetic relationship, cohesins have been separated into three distinct types. By definition, the dockerins that interact with each type of cohesin are of the same type.

 

Although structurally related, type I cohesins and dockerins were shown to be different from type II (15-25% identity) and do not cross react. In fact, comparison of the primary structure of C. thermocellum cohesins and dockerins shows small degree of similarity between them, consistent with the lack of cross-specificity between type I and type II cohesin–dockerin pairs. Several studies shown that type I cohesins of C. thermocellum recognise almost all of type I dockerins present on the enzymatic subunits but, interestingly, type I and type II cohesin/dockerins partners do not interact, ensuring a clear distinction between the mechanism for cellulosome assembly and cell-surface attachment. Furthermore, it was also shown that, although type I cohesins/dockerins from one species do not interact with other type I cohesins/dockerins from other species, type II cohesins/dockerins demonstrate a rather extensive cross-species plasticity. The biological relevance of this cross-species interaction is still uncertain. The fact that type I cohesins in the enzymatic units recognise nearly all the type I dockerins in the scaffoldin unit suggests that, within a given species, the arrangement of the several enzymes occurs randomly along the scaffoldin, reflecting, perhaps, the complexity of the substrate in which the microbe is.

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Carbohydrate-Binding Modules (CBMs)​

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As mentioned, in order to degrade the highly complex plant cell wall, microorganisms have developed a specialised complex (cellulosome) composed by multiple enzymes and non-catalytic modules. Many carbohydrate-active enzymes are modular protein bound to one or more non-catalytic carbohydrate-binding modules (CBMs) that function in an independent manner.

 

A CBM is defined as a continuous amino acid sequence within a carbohydrate-active enzyme with a separate fold having carbohydrate-binding activity. To date several hundred putative CBM sequences have been identified experimentally and classified into different families according to their sequence similarity (Carbohydrate Active Enzymes database - http://www.cazy.org). CBMs are composed by 30 to 200 amino acids and they occur as a single, double or triple domain in one protein. They can be found at the C- or N-terminal of the catalytic protein and, invariably, their key role is to recognise and specifically bind to the several different carbohydrates found in the plant cell wall. This specific recognition and binding to the carbohydrates of the plan cell wall has considerable biological consequences such as:

 

• Anchoring the multienzyme complex to the substrate;

• Bringing the catalytic domain in close proximity to the substrate and, therefore, enhancing the hydrolysis of insoluble substrates through an effective increase of the concentration of cellulase on the surface of the substrate;

• Disrupting the structure of the polysaccharides.

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| "...their key role is to recognise and specifically bind to the several different carbohydrates."

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Our knowledge on these systems as grown considerably over the last years as a result of structural information provided by NMR spectroscopic and X-ray crystallographic studies deepening our understanding on the biological functions of CBMs. In addition to plant cell wall carbohydrate recognition, CBMs are involved in a large number of other processes such, pathogen defense, polysaccharide biosynthesis, virulence, plant development, etc.

 

Therefore, understanding of the CBMs properties and mechanisms of ligand binding and recognition is imperative for the development of new carbohydrate-recognition technologies and for providing the basis for fine manipulation of the carbohydrate–CBM interactions.

 

 

 

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