Carbohydrate-binding modules

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• Author: ^^^Alicia Lammerts van Bueren^^^
• Responsible Curator: ^^^Al Boraston^^^ and ^^^Spencer Williams^^^

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This page is under construction. In the meantime, please see these references for an essential introduction to the CAZy classification system: [1, 2]. CBMs, in particular, have been extensively reviewed[3, 4, 5, 6].

Overview

Carbohydrate-binding modules (CBMs) are defined as a stretch of amino acid sequence within a larger encoded protein sequence and folds into a discreet and independent module, forming part of a larger multi-modular protein. Most commonly associated with glycoside hydrolases (but also polysaccharide lyases, polysaccharide oxidases, glycosyltransferases and expansins), their role is to bind to carbohydrate ligand and direct the catalytic machinery onto its substrate, thus enhancing the catalytic efficiency of the multimodular carbohydrate-active enzyme. CBMs are themselves devoid of any catalytic activity.

Insert here a classical example demonstrating "modularity" of a CAZyme with CBMs

History of CBMs

CBMs were initially characterized as cellulose binding domains in cellobiohydrolases CBHI and CBHII from Trichoderma reesei [7, 8] and cellulases CenA and CexA from Cellulomonas fimi [9]. Limited proteolysis experiments of these enzymes yielded truncated enzyme products that showed a reduced or complete loss in their ability to hydrolyze cellulose substrates. The reduction in enzymatic activity was attributed to the loss of ~100 amino acid C-terminal domains which prevented the adsorbption of the enzymes onto cellulose substrate. Thus it was proposed that these independent "cellulose binding domains" are critical for targeting the enzymes onto its substrate and enhancing their hydrolytic activity.

Classification

Sequence Based Classification

Carbohydrate-binding modules are currently classified into 67 families based on amino acid sequence similarities (May 2013), which are available through the Carbohydrate Active enZyme database. Sequence-based relationships often cluster together modules with similar structural folds and carbohydrate-binding function. While this is true for most CBM families, there are several families that exhibit diversity in the carbohydrate ligands they target (examples include CBM6, CBM32, others...)

Fold

Structural information for over 90% of the CBM families is known. The most common fold exhibited by CBMs is the beta-sandwich fold which is comprised of two overlapping beta-sheets consisting of 3 - 6 antiparallel beta strands. The binding sites for carbohydrates have been shown to be located either on the same face of a beta-sheet (ref), on the edge of the beta-sheet within the joining loops (ref), or both (ref)

Picture

Other folds include the beta-trefoil fold, cysteine knot, OB fold, the hevein and hevein-like folds and unique [3]

Types

CBMs are static molecules as they do not undergo any conformational changes when binding to their target ligand. Because CBMs do not undergo conformational changes, the topography of their binding site is "preformed" to be complementary to the shape of the target carbohydrate ligand. Amino acid side chains within the CBM binding pocket create a complementary "platform" that is optimally suited to bind the target ligand. This feature allows CBMs to be classified into three main Types that are defined by the shape and degree of polymerization of their ligand target.

Type A: polycrystalline surface binding

Type B: oligosaccharides with DP>4 (mainly endo, within polysaccharide chains)

Type C: lectin-like mono/di/tri saccharides (mainly exo, reducing/non-reducing end)

Properties of Carbohydrate Binding Interactions

There are two key forces which drive CBM/carbohydrate interactions. Hydrogen bonds and hydrophobic stacking interactions.

Roles of CBMs

The association of CBMs with Glycoside hydrolases (and other CAZymes) and their ability to interact with unique positions within polysaccharides conveys four main functional roles to the activity of the associated catalytic module:

Targeting

Proximity

Cell Wall anchoring. CBM35 modules have been shown to interact with the surface glucuronic acid containing sugars in Amycolatopsis orientalis

Disruptive role has previously been described for cellulose binding CBM2a. Additionally starch binding CBM20 may have a disruptive role in amylose while dual-associated CBM41 modules may have a disruptive role in degrading glycogen granules. CBM33 was thought to have a disruptive effect on chitin, however these have now been reclassified as lytic oxygenases (expand).

Defining a new CBM family

In order to define a new CBM family, one must:

1. Demonstrate an independent module as part of a larger carbohydrate-active enzyme.

2. Demonstrate binding to carbohydrate ligand.

3. Additional family members are then determined based on amino acid sequence similarity. To be defined as a true CBM, it must form part of a larger amino acid sequence encoding a putative CAZyme (or enzyme with demonstrated activity on a carbohydrate-containing substrate and the CBM enhances the catalytic efficiency of the enzyme by binding with or in close proximity of the substrate).

Amino acid sequence-based classification of a CBM family may lead to the incorporation of other carbohydrate binding proteins within a given family, including lectins (such as ricin (CBM13), tachycitin (CBM14), wheat germ agglutinin (CBM18), fucolectin (CBM47), and malectin (CBM57)) and periplasmic solute binding proteins (such as CBM32). However to meet the true definition of a CBM, all of the above three mentioned criteria must be met.

References

1. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. Biochem. J. (BJ Classic Paper, online only). DOI: 10.1042/BJ20080382

2. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |
3. Boraston AB, Bolam DN, Gilbert HJ, and Davies GJ. (2004). Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J. 2004;382(Pt 3):769-81. DOI:10.1042/BJ20040892 |
4. Hashimoto H (2006). Recent structural studies of carbohydrate-binding modules. Cell Mol Life Sci. 2006;63(24):2954-67. DOI:10.1007/s00018-006-6195-3 |
5. Shoseyov O, Shani Z, and Levy I. (2006). Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev. 2006;70(2):283-95. DOI:10.1128/MMBR.00028-05 |
6. Guillén D, Sánchez S, and Rodríguez-Sanoja R. (2010). Carbohydrate-binding domains: multiplicity of biological roles. Appl Microbiol Biotechnol. 2010;85(5):1241-9. DOI:10.1007/s00253-009-2331-y |

7. Van Tilbeurgh, H., Tomme P., Claeyssens M., Bhikhabhai R., Pettersson G.(1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei. FEBS Lett. 204,223–227. [1]

8. Tomme P, Van Tilbeurgh H, Pettersson G, Van Damme J, Vandekerckhove J, Knowles J, Teeri T, and Claeyssens M. (1988). Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem. 1988;170(3):575-81. DOI:10.1111/j.1432-1033.1988.tb13736.x |
9. Gilkes NR, Warren RA, Miller RC Jr, and Kilburn DG. (1988). Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem. 1988;263(21):10401-7. | Google Books | Open Library