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6.3b: Composition of Enzymes

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  • 6.3b Composition of Enzymes

    The first place to start is to describe the structure of a cellulase using typical terms in biochemistry. A modular cellobiohydriolase (CBH) has a few aspects in common; the common features include: 1) binder region of the protein, 2) catalytic region of the protein, and 3) a linker region that connects the binder and catalytic regions. Figure 6.14a shows a general diagram of the common features of a cellulase. The CBH is acting on the terminal end of a crystalline cellulosic substrate, where the cellulose binding domain (CBD) is imbedded in the cellulose chain, and the strand of cellulose is being digested by the enzyme catalyst domain to produce cellobiose. This type of enzyme is typical of exocellulases. Figure 6.14b shows a more realistic model, where the linker is attached to the surface of the cellulose.

    One of the main differences between glycosyl hydrolases (a type of cellulase) and the other enzymes how the catalytic domain functions. There are three types: 1) pocket, 2) cleft, and 3) tunnel. Pocket or crater topology (Figure 15A) is optimal for the recognition of a saccharide non-reducing extremity and is encountered in monosaccharidases. Exopolysaccharidases are adapted to substrates having a large number of available chain ends, such as starch. On the other hand, these enzymes are not very efficient for fibrous substrates such as cellulose, which has almost no free chain ends. Cleft or groove cellulase catalytic domains are “open” structures (15B), which allow a random binding of several sugar units in polymeric substrates and is commonly found in endo-acting polysaccharidases such as endocellulases. Tunnel topology (Figure 6.15C) arises from the previous one when the protein evolves long loops that cover part of the cleft. Found so far only in CBH, the resulting tunnel enables a polysaccharide chain to be threaded through it. The red portions on each catalytic domain is supposed to be the carbohydrate being processed, although it is difficult to see in this picture.

    CMH attacks crystalline cellulose It has a cellulose binder domain, linker region doing glycosylation & catalytic domain yielding cellobiose
    Figure 6.14a: Biochemistry of cellulases – a modular cellobiohydriolase (CBH).

    Credit: Himmel et al., Cellulases: Structure, Function, and Applications. In: Handbook on Bioethanol: Production and Utilization.

    Model schematic of what cellulases look like
    Figure 6.14b: Model schematic of what cellulases look like, showing a more realistic picture of the catalytic domain, binding module, and linker.

    Credit: National Renewable Energy Laboratory

    Catalytic Domains of Glycosyl Hydrolases – A) pocket, B) cleft, and C) tunnel, see text description in link below
    Figure 6.15: Catalytic Domains of Glycosyl Hydrolases – A) pocket, B) cleft, and C) tunnel.

    Click for a text description of Figure 6.15

    Catalytic Domains of Glycosyl Hydrolases:

    Pocket - glucomylase from A. awamori, Hydrolysis of amorphous polymers or dimers (e.g. starch and cellobiose)

    Cleft - Endoglucanase (Cel6A) from T. fusca Hydrolysis of crystalline polymers (e.g. cellulose endoglucanases)

    Tunnel - exoglucanase CHBII (Cel6A) from T. reesei, processive hydrolysis of crystalline polymers (e.g. exoglucanases)

    Credit: Davies and Henrissat, 1995

    The other main feature of these enzymes is the cellulose binding domain or module (CBD or CBM). Different CBDs target different sites on the surface of the cellulose; this part of the enzyme will recognize specific sites, help to bring the catalytic domain close to the cellulose, and pull the strand of cellulose molecule out of the sheet so the glycosidic bond is accessible.

    So now, let’s go back to noncomplexed versus complexed cellulase systems. Figure 6.16 is another comparison of noncomplexed versus complexed cellulase systems, but this time, it focuses on the enzymes. Notice in Figure 6.16A, the little PacMan look-alike figures for enzymes. The enzymes are separate, but work in concert to break down the cellulose strands into cellobiose and glucose. Recall that this process is aerobic (in oxygen).

    Now look at Figure 6.16B and the complexed system. The enzymes are attached to subunits that are attached to the bacterium cell wall. The products are the same, but recall that this system in anaerobic (without oxygen), and these enzymes all work together to produce cellobiose and glucose.

    Noncomplexed versus complexed cellulase systems. Described in text above
    Figure 6.16: Noncomplexed versus complexed cellulase systems, focusing on the enzymes and their differences in both systems.

    Credit: Khanok Ratanakhanokchai, Rattiya Waeonukul,
    Patthra Pason, Chakrit Tachaapaikoon, Khin Lay Kyu,
    Kazuo Sakka, Akihiko Kosugi and Yutaka Mori (2013). Paenibacillus curdlanolyticus Strain B-6 Multienzyme Complex: A Novel System for Biomass Utilization, Biomass Now - Cultivation and Utilization, Dr. Miodrag Darko Matovic (Ed.), ISBN: 978-953-51-1106-1, InTech, DOI: 10.5772/51820.

    So, what are those subunits that are essentially the connectors in the enzyme? Figure 6.17 shows a schematic of the types. The cellulosome is designed for the efficient degradation of cellulose. A scaffoldin subunit has at least one cohesin modules that are connected to other types of functional modules. The CBM shown is a cellulose-binding module that helps the unit anchor to the cellulose. The cohesin modules are major building blocks within the scaffoldin; cohesins are responsible for organizing the cellulolytic subunits into the multi-enzyme complex. Dockerin modules anchor catalytic enzymes to the scaffoldin. The catalytic subunits contain dockerin modules; these serve to incorporate catalytic modules into the cellulosome complex. This is the architecture of the C. thermocellum cellulosome system. (Alber et al., CAZpedia, 2010). Within each cellulosome, there can be many different types of these building blocks. Figure 6.18 shows a block diagram of two different structures of T. neapolitana LamA and Caldicellulosiruptor strain Rt8B.4 ManA in a block diagram form. Due to the level of this class, we will not be going into any greater depth about these enzymes.

    Cellulosome components for complexed enzyme, described in text above
    Figure 6.17: Cellulosome components for a complexed enzyme.

    Credit: Cellusome: from CAZYpedia

    Modular structure of T. neapolitana LamA and Caldicellulosiruptor strain Rt8B.4 ManA. Shows peptides in their sequence
    Figure 6.18: Modular structure of T. neapolitana LamA and Caldicellulosiruptor strain Rt8B.4 ManA.

    Credit: Summa et al., Biochem. J., 2001

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