Structural Biochemistry/Cell Organelles/Plant Cell/Cellulosomes

Overview
Cellulosomes are a complex of multi-cellulolytic enzymes that break down cellulose and hemicelluloses found in the cell walls of plants. Cellulose and hemicelluloses are two organic polysaccharide compounds that are also two of the most abundant polymers on Earth. They are major sources of carbon and energy. Cellulases and hemicellulases participate in protein:carbohydrate [carbohydrate-binding modules (CBMs)] or protein:protein (dockerins) interactions that make it so hard to break down. Chemically, cellulose is relatively simple. However its crystalline structure makes it resistant to biological degradation. Deconstruction and digestion of cellulose and hemicelluloses will allow for carbon turnover and numerous biotechnological uses, including biofuels as an alternative fuel source. However, very few microorganisms have the capacity to digest cellulose and hemicelluloses, which are why cellulosomes are of great interest.

Discovery
The first cellulosome was discovered in the early 1980s by Bayer and Lamed in their studies of the cellulolytic system of Closteridium thermocellum, an anaerobic bacterium. They described the cellulosome as a “discrete, cellulose-binding, multi-enzyme complex that mediates the degradation of cellulosic substrates.” They initially believed that the cellulosome only degraded cellulose, but it was later recognized that the cellulosome also degrade a large number of hemicellulases and even pectinases. Also following Bayer and Lamed’s studies were research in identifying the molecular mechanisms of how the enzyme complex assembles and how the cellulosome is present on the surface of the host bacterium. More recently, a range of anaerobic bacteria and fungi were shown to also produce cellulosome systems.

Function
Scientists have argued that cellulosomes are more efficient in digesting cellulose and hemicellulases than corresponding enzyme systems produced by aerobic bacteria and fungi. It is possible that anaerobic environments imposed selective pressures that led to the evolution of cellulosomes. One hypothesis proposes that the integration of plant cell wall-degrading enzymes onto a macromolecular complex leads to a more efficient enzyme complex whose cellulosomal catalytic units interactions work together synergistically and further enhanced by enzyme substrate targeting through scaffoldin-borne CBM. Another hypothesis suggest that the function of the cellulosome on the surface of the bacterium, at least in C. thermocellum, enhances the capacity of the host bacterium to utilize mono- and oligosaccharide released from the cell wall.

General Structure
The cellulosome was found to be composed cellulosomal catalytic components that contained noncatalytic modules called dockerins, which bind to cohesin modules. This receptor/adapter protein domain pair is responsible for cellulosome self-assembly. The recognition between cohesin and dockerin is type and/or species specific. Together cohesin and dockerin are located in a large noncatalytic protein that acts as a scaffoldin.

Dockerins
Dockerins consists of approximately 70 amino acids that contain two segments, each of about 22 residues, in the cellulosomal catalytic components. The first 12 residues resemble the calcium-binding loop of EF hand motifs in which aspartate or asparagines are highly conserved. The EF hand is a helix-loop-helix structural domain found in a large family of calcium-binding proteins. Calcium was shown to be necessary for dockerin stability and function. Without calcium, dockerins are unable to interact with cohesions. They are also present in a single copy at the C-terminus of cellulosomal enzymes.

Cohesins and Scaffoldins
Cohesins are 150 residue modules of tandem repeats in scaffoldins, which specifically bind to dockerin modules. The number of cohesin modules in scaffoldins varies between one and eleven, but is usually more than four. Scaffoldins can be defined as cohesin containing proteins, which play a role in cellulosome assembly. The scaffoldin subunits of the cellulosome function to organize and position other protein subunits into the complex and can also serve as an attachment device for harnessing the cellulosome onto the cell surface and/or for targeting its substrate because scaffoldins usually contain a noncatalytic CBM that anchors the entire complex onto cellulose. More recently, researchers found that some cellulosome-producing microbes produce more than one type of scaffoldin. Scaffoldins that bind cellulases and hemicellulases are called primary scaffoldins. Proteins that bind primary scaffoldins are called anchoring scaffoldins.

How Cellulosomes Bind to Plant Cell Walls
Cell-surface attachment of cellulosomes is required for degradation of plant cell wall polysaccharides. CBMs are carbohydrate-binding modules that are separated into three types: Type A (interacts with crystalline polysaccharides), Type B (bind to internal regions of single glycan chains), and Type C (recognizes small saccharides). It was first thought that bacterial cellulosomes are bound to the cellulose by the “cellulose-binding factor,” but it was later realized that the attachment of the cellulosome to the cell wall is mediated by a family 3 CBM (CBM3) located in the scaffoldins. CBM3s are type A modules that bind tightly to the cellulose surface.

Bacterial Cellulosomes
There are two major types of bacterial cellulosomes: those present in multiple types of scaffoldins and those that contain a single scaffoldin. Cellulosomes that assemble via a single primary scaffoldin are the simplest and contain six to nine catalytic components, depending on the number of cohesions in the primary scaffoldin. Bacterial cellulosomes are bound tightly to the cellulose and referred to as the “cellulose-binding factor.” Bacteria expressing cell-surface cellulosomes contain a single primary scaffoldin and multiple anchoring scaffoldins. The majority of the anchoring scaffoldins contain SLH modules or SLH domains, which mediate the attachment of the structural proteins to the bacterial cell wall and may bind to the secondary cell wall polysaccharides.

Fungal Cellulosomes
Anaerobic fungi can hydrolyze many different polysaccharides, including cellulose and hemicelluloses. It is believed that anaerobic fungi are the initial invader of lignocelluloses and play a role in fiber digestion along with bacteria and other microorganisms. Anaerobic fungal plants have cellulosomes just like anaerobic bacteria. However, fungal cellulosomes are less characterized. A number of dockerin sequences have been identified in different fungi strains. Fungal dockerins consist of a three-stranded beta-sheet and a short helix held together by two disulfides. Researchers also found that the amino acid sequences of fungal dockerins are unrelated to their bacterial counterparts. Genome sequencing of fungi will allow scientists to see which dockerin sequences present in proteins are associated with plant wall degradation. Another difference between fungal and bacterial cellulosomes is that fungal cellulosomes generally contain two copies of dockerin modules, each consisting of approximately 40 residues joined together by shorter linker sequences. Researchers have also hypothesized that fungal cellulosome may not involve cohesin modules. However, more work and research is needed in characterizing fungal cellulosomes.

Applications
Studying cellulosomes and how they work would provide a large number of biotechnical benefits to society. There are many industrial applications, such as paper pulp production, that requires breakdown of plant cell walls in its process. Cellulosomes would be a great benefactor. One of the biggest challenges facing the world today is the lack of alternative and renewable energy sources to the conventional fossil fuels. A potential, viable alternative can be found in the converting lignocellulosic biomass to fermentable sugars to produce renewable fuels, such as ethanol. Yet, the biggest rate limiting problems in converting lignocelluloses into biofuels, is the hydrolysis of structural polysaccharides, which require the development of a more efficient enzyme system. Extensive enzyme group is needed to overcome the recalcitrant and the plant cell wall. Cellulosomes could be the future solution.