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Carbohydrate-binding modules
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- Author: ^^^Alicia Lammerts van Bueren^^^
- Responsible Curator: ^^^Al Boraston^^^ and ^^^Spencer Williams^^^
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 fold into a discreet and independent module, forming part of a larger multi-modular protein (Figure 1). The role of a CBM 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. CBMs are most commonly associated with Glycoside Hydrolases but have also been identified in Polysaccharide Lyases, polysaccharide oxidases, Glycosyltransferases and plant cell wall-binding expansins [8].
CBMs themselves do not undergo any conformational changes when binding ligand. Rather, the topography of the carbohydrate-binding site is preformed to be complementary to the shape of the target ligand (see Types). This is achieved by the presence of amino acid side chains and loops within the CBM binding pocket. However multimodular enzymes as a whole may be quite flexible and experience complete conformational changes when binding substrate. Flexible loop regions between adjacent modules can allow for shifts in the orientation and direction of the catalytic module with respect to the CBM on the target substrate [9].
History of CBMs
CBMs were initially characterized as cellulose binding domains (CBDs) in cellobiohydrolases CBHI and CBHII from Trichoderma reesei [10, 11] and cellulases CenA and CexA from Cellulomonas fimi [12]. 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 "domains" are critical for targeting the enzymes onto its substrate and enhancing their hydrolytic activity.
CBDs were previously categorized into 13 Types based on amino acid sequence similarities [13]. This classification system became complicated when similar functional domains from non-cellulolytic carbohydrate-active enzymes were discovered that did not bind cellulose but met all of the criteria of a CBD. The term carbohydrate-binding module was proposed to solve this problem to be inclusive of all ancillary modules with non-catalytic carbohydrate-binding function (for a review see [3]).
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)
Fold
Structural information for 54 of the 67 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 three to six antiparallel beta strands (Figure 2). The ligand binding site is located primarily on the same face of a beta-sheet (Figure 2A), but may also be positioned on the edge of the beta-sheet within the joining loop region (Figure 2B). There are examples of CBMs exhibiting dual binding sites [16]. Other folds include the beta-trefoil fold, cysteine knot, OB fold, the hevein and hevein-like and unique folds [3].
Types
CBMs are classified into three main Types defined by the shape and degree of polymerization of their target ligand (Figure 3A). The architecture of the binding site determines what region within a polysaccharide the enzyme will target. A recent review on CBM plant cell wall recognition [18] has modified the classification of CBM Types to be as follows:
- Type A: bind to crystalline surfaces of cellulose and chitin (example families CBM1, CBM2, CBM3, CBM5, CBM10). Their binding sites are planar and rich in aromatic amino acid residues creating a flat platform to bind to the flat polycrystalline chitin/cellulose surface (Figure 3B). Type A CBMs are unique and differ significantly from Type B or C.
- Type B: bind internal glycan chains (endo-type). The most abundant CBM type. Type B binding sites appear as extended grooves or clefts comprised of binding subsites to accommodate longer sugar chains with four or more monosaccharide units.
- Type C: bind termini of glycans (reducing/non-reducing ends, exo-type). Type C binding sites are short pockets for recognizing short sugar ligands containing one to three monosaccharide units (example families CBM9, CBM13, CBM47). Families containing Type C CBMs often include lectins as members.
Properties of Carbohydrate Binding Interactions
Functional Roles of CBMs
CBMs carry out four main functional roles:
Targeting Effect: CBMs target the enzyme to distinct regions within a larger macromolecular polysaccharide substrate (reducing end, non-reducing end, internal polysaccharide chains), depending on the architecture of its binding site (see Types).
Proximity Effect: CBMs increase the concentration of enzyme in close proximity to its polysaccharide substrate. This leads to more rapid and efficient substrate degradation.
Disruptive Effect: Some CBMs have been shown to disrupt the surface of tightly packed polysaccharides, such as cellulose fibres and starch granules, causing the substrate to loosen and become more exposed to the catalytic module for more efficient degradation. Disruptive roles have been described for cellulose binding CBM2a [19] and CBM44 [20]. 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 Copper-dependent lytic polysaccharide monooxygenases [21] and are reclassified as Auxillary Activity Family 9.
Adhesion: CBMs have been shown to adhere enzymes onto the surface of bacterial cell wall components while exhibiting catalytic activity on an external neighboring carbohydrate substrate. For example, CBM35 modules have been shown to interact with the surface glucuronic acid containing sugars in the cell wall of Amycolatopsis orientalis while the catalytic module is active on external chitosan likely originating from the cell wall of competing soil fungal species [22].
Driving Forces of CBM/Carbohydrate Interactions
There are two key features that drive CBM/carbohydrate interactions. Extensive hydrogen bonding occurs between the hydroxyl groups of carbohydrate ligands and polar amino acid residues within the binding site. Additional water-mediated hydrogen bonding networks between these groups can also be found in the binding site. By far the most important characteristic driving force mediating protein-carbohydrate interactions is the position and orientation of aromatic amino acid residues (Try, Tyr and sometimes Phe) within the binding site. These essential planar residues form hydrophobic stacking interactions with the planar face of sugar rings. Weak intermolecular electrostatic interactions occur between C-H and pi electrons in the planar ring systems and contribute 1.5 - 2.5 kcal/mol energy to the binding reaction [23].
Metal binding sites...
CBM Promiscuity
Because of the diversity of carbohydrate structures and motifs found in plant and mammalian glycans, some CBMs feature promiscuity in ligand recognition. While the core monosaccharide in the primary subsite remains important for initial recognition of carbohydrate ligand, CBMs may exhibit flexibility in what sugar monomers can be accommodated in binding subsites. Examples include...
CBMs and Multivalency
CBMs and Lectins
Several lectins [24, 25] fall into the CBM classification system in the Carbohydrate Active enZyme database as they exhibit similar folds and carbohydrate binding properties with CBMs. For example, ricin toxin B chain from Ricinus communis resides in family CBM13, while wheat germ agglutinin (WGA) can be found in family CBM18. The human lectin malectin is classified as family CBM57 and plays a role in N-linked glycan processing of polypeptides in the endoplasmic reticulum [26]. CBMs also share properties with lectins that are not yet incorporated in the Carbohydrate Active enZyme database. For example the fucose-specific Anquila anguila lectin AAA is similar to Type C CBMs found in family CBM6 and CBM32 [15]. Lectins which are classified as CBMs are incorporated into a family because they were found to share amino acid sequence identity with a known CBM appended to a carbohydrate-active enzyme (see criteria).
The most prominent feature that separates CBMs from lectins is the involvement of lectins in agglutination of sugar-containing molecules or glycoconjugates. Lectins often form quaternary structures as homodimers, trimers or tetramers with several binding sites which then agglutinate the target glycocongugate [24, 25]. CBMs themselves are not involved in the formation of quaternary structures and do not have agglutinating properties.
Lectins are also thought to have a higher density of hydrogen bond formation with their carbohydrate ligand than CBMs and may differ in their ability to utilize thermodynamic contributions from solvent rearrangement during ligand binding (references).
An excellent, brief historical overview of the discovery and characterization of lectins is available [24].
Criteria for Defining a new CBM family
In order to define a new CBM family, one must:
- Demonstrate an independent module as part of a larger carbohydrate-active enzyme.
- Demonstrate binding to carbohydrate ligand.
- 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). The community is open to incorporation of all carbohydrate-binding proteins within the CBM classification system based on the above criteria.
Studying CBM-ligand Interactions
Laboratory approaches to studying the binding function of carbohydrate-binding modules has been extensively reviewed [27]. Typically, molecular biology techniques are used to overproduce a CBM protein in a host strain such as Escherichia coli which is then isolated and purified. Initial screening for carbohydrate binding interactions can be performed using screening techniques such as microarrays [28] or fluorescence microscopy techniques [28, 29, 30]. Several approaches can be taken to verify and quantify CBM-polysaccharide interaction, including affinity gel electrophoresis, UV difference and fluorescence spectroscopy, solid state depletion assay and isothermal titration calorimetry [31].
Overall demonstration of carbohydrate binding function by CBMs is essential to understanding how these associated modules confer enzymatic specificity to carbohydrate-active enzymes. Features of CBMs are currently being exploited to create designer CAZymes with enhanced or modified carbohydrate recognition functions [32].
References
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