Carbohydrate-binding modules - Revision history

Elizabeth Ficko-Blean: /* Functional Roles of CBMs */

2023-01-09T09:52:27Z

Functional Roles of CBMs

Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

2021-12-18T21:14:24Z

Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

Shinya Fushinobu: fixing typos

2020-07-08T04:06:10Z

fixing typos

Elizabeth Ficko-Blean at 09:58, 30 August 2018

2018-08-30T09:58:52Z

Spencer Williams at 15:30, 23 May 2018

2018-05-23T15:30:26Z

Elizabeth Ficko-Blean at 20:28, 2 May 2018

2018-05-02T20:28:17Z

Elizabeth Ficko-Blean at 20:26, 2 May 2018

2018-05-02T20:26:10Z

Elizabeth Ficko-Blean at 16:27, 1 May 2018

2018-05-01T16:27:11Z

Elizabeth Ficko-Blean at 16:26, 1 May 2018

2018-05-01T16:26:13Z

Elizabeth Ficko-Blean at 15:46, 1 May 2018

2018-05-01T15:46:12Z

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 *''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 <cite>Montanier2009</cite>. ''Streptococcus pneumoniae'' uses a [[CBM71]] as an adhesin, to mediate adherence to host cell surfaces displaying lactose or N-acetyllactosamine <cite>king2014</cite>.
-There are two examples in the literature of CBMs extending the active site sub-sites of their appended glycosidase modules.  The glycogen-degrading pneumococcal virulence factor SpuA has its active site extended by one of two tandem [[CBM41]]s <cite>Lammerts2011</cite>. The glucan starch phosphatase Starch Excess4 has its active site extended by a [[CBM48]] <cite>Meekins2014</cite>.
+There are examples in the literature of CBMs extending the active site sub-sites of their appended glycosidase modules.  The glycogen-degrading pneumococcal virulence factor SpuA has its active site extended by one of two tandem [[CBM41]]s <cite>Lammerts2011</cite>. The glucan starch phosphatase Starch Excess4 has its active site extended by a [[CBM48]] <cite>Meekins2014</cite>.
 Several lectins are classified as CBMs even though they are not on the same polypeptide chain as a carbohydrate-active enzyme.  See [[Carbohydrate-binding_modules#CBMs, Lectins and Outliers|Blurred Lines: CBMs, Lectins and Outliers]] for a more complete discussion.
 ===Driving Forces of CBM-Carbohydrate Interactions===

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 {{CuratorApproved}}
-* [[Author]]: ^^^Alicia Lammerts van Bueren^^^ and ^^^Elizabeth Ficko-Blean^^^
+* [[Author]]: [[User:Alicia Lammerts van Bueren|Alicia Lammerts van Bueren]] and [[User:Elizabeth Ficko-Blean|Elizabeth Ficko-Blean]]
-* [[Responsible Curator]]:  ^^^Al Boraston^^^ and ^^^Spencer Williams^^^
+* [[Responsible Curator]]:  [[User:Al Boraston|Al Boraston]] and [[User:Spencer Williams|Spencer Williams]]
 ----

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 === Fold ===
 [[Image:CBMfold.jpg|thumb|right|500px|'''Figure 2. Classical CBM beta-sandwich fold.''' C-terminal family CBM27 from ''Thermotoga maritima'' mannanase, a Type B CBM (A)(side and front view, PDB ID [{{PDBlink}}1OF4 1OF4]) <cite>Boraston20031</cite> and C-terminal family CBM6 from ''Clostridium stercorarium'' xylanase (B) (PDB ID [{{PDBlink}}1NAE 1NAE]) <cite>Boraston20032</cite> showing binding sites on the face (A) and loop region (B) of the beta sandwich fold respectively.]]
-CBMs fall into one of 7 fold families <cite>Boraston2004</cite>. The most common fold exhibited by CBMs is the beta-sandwich fold comprised of two overlapping beta-sheets each consisting of three to six antiparallel beta strands (Figure 2). The ligand binding site may be located on one face of the beta-sheet (Figure 2A) or may be positioned within the variable loop region of the beta-sheet (Figure 2B). There are examples of CBMs in the beta-sandwich fold family exhibiting dual binding sites such as [[CBM6]] <cite>Pires2004</cite> and dual starch-binding sites in [[CBM20]] <cite>Lawson1994</cite>. Other fold families include the beta-trefoil fold, cysteine knot, OB fold, the hevein and hevein-like and unique folds <cite>Boraston2004</cite>. CBMs of the beta-trefoil fold family ([[CBM13]], [[CBM42]]) present multivalent sugar-binding sites, as demonstrated for their interaction with xylan and arabinoxylan respectively <cite>Fujimoto2013</cite>.
+CBMs fall into one of 7 fold families <cite>Boraston2004</cite>. The most common fold exhibited by CBMs is the beta-sandwich fold comprised of two overlapping beta-sheets each consisting of three to six antiparallel beta strands (Figure 2). The ligand binding site may be located on one face of the beta-sheet (Figure 2A) or may be positioned within the variable loop region of the beta-sheet (Figure 2B). There are examples of CBMs in the beta-sandwich fold family exhibiting dual binding sites such as [[CBM6]] <cite>Pires2004</cite> and dual starch-binding sites in [[CBM20]] <cite>Lawson1994</cite>. Other fold families include the beta-trefoil fold, cystine knot, OB fold, the hevein and hevein-like and unique folds <cite>Boraston2004</cite>. CBMs of the beta-trefoil fold family ([[CBM13]], [[CBM42]]) present multivalent sugar-binding sites, as demonstrated for their interaction with xylan and arabinoxylan respectively <cite>Fujimoto2013</cite>.
 === Types ===

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 *''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 [[CBM2]]a <cite>Din1991</cite> and [[CBM44]] <cite>Gourlay2012</cite>. Dual starch-binding domains of family [[CBM20]] from ''Aspergillus niger'' glucoamylase have been shown to disrupt the surface of starch <cite>Southall1999</cite> while dual-associated [[CBM41]] modules may have a disruptive role in degrading glycogen granules <cite>vanBueren2007</cite>. [[CBM33]] was thought to have a disruptive effect on chitin, however these have now been reclassified as copper-dependent lytic polysaccharide monooxygenases <cite>Vaaje2010</cite> and are found in CAZy [[Auxiliary Activity Family 10]].
-*''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 <cite>Montanier2009</cite>.
+*''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 <cite>Montanier2009</cite>. ''Streptococcus pneumoniae'' uses a [[CBM71]] as an adhesin, to mediate adherence to host cell surfaces displaying lactose or N-acetyllactosamine <cite>king2014</cite>.
 There are two examples in the literature of CBMs extending the active site sub-sites of their appended glycosidase modules.  The glycogen-degrading pneumococcal virulence factor SpuA has its active site extended by one of two tandem [[CBM41]]s <cite>Lammerts2011</cite>. The glucan starch phosphatase Starch Excess4 has its active site extended by a [[CBM48]] <cite>Meekins2014</cite>.
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 #Jervis1997 pmid=9295354
 #Ficko-Blean2006 pmid=16990278
 </biblio>
 [[Category:Definitions and explanations]]

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-<!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
+{{CuratorApproved}}
 * [[Author]]: ^^^Alicia Lammerts van Bueren^^^ and ^^^Elizabeth Ficko-Blean^^^
 * [[Responsible Curator]]:  ^^^Al Boraston^^^ and ^^^Spencer Williams^^^

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 The biological reaction of agglutination is when particles that are suspended in a liquid collect into clumps, such as that occuring as a serologic response to a specific antibody. The most prominent feature that is genarally considered to separate CBMs from lectins is the involvement of lectins in agglutination of sugar-containing molecules or glycoconjugates.  Lectins exploit multivalency, often forming quaternary structures as homodimers, trimers or tetramers with several binding sites which then agglutinate the target glycocongugate <cite>SharonLis2004 SharonLis2007</cite>. Few studies have been done on the agglutinating effects of CBMs or CBM tandems; however, a [[CBM26]]/[[CBM25]] pair from ''Bacillus halodurans'' is described as strongly agglutinating on soluble amylopectin (and pullulan), suggesting multivalent binding of the individual CBMs to sites on separate glucan chains <cite>Boraston2006</cite>. CBMs individually are not known to be directly involved in the formation of quaternary structures and are not known to have agglutinating properties - in common with sugar-recognition modules of all glycan-binding proteins, including lectins <cite>Taylor2014</cite>. Other examples of CBMs participating in quaternary structures but not directly implicated in quaternary structure formation are found in cellulosome complexes <cite>Freelove2001 Poole1992 Morag1995</cite> and in some secreted pathogenic bacterial enzymes complexes <cite>Adams2008 Ficko2009</cite> where complex formation is mediated through specific cohesin-dockerin module interactions.
-Amino acid sequence-based classification of a CBM family may lead to the incorporation of other non-catalytic-associated CBMs within a given family. Some examples of families containing CBMs without appended catalytic modules include those with lectins (such as tachycitin ([[CBM14]]), wheat germ agglutinin ([[CBM18]]), fucolectin ([[CBM47]]), and malectin ([[CBM57]])), and those with periplasmic solute binding proteins (such as within [[CBM32]]). Interestingly, the lectin ricin B chain ([[CBM13]]), while not on the same polypeptide chain, is covalently linked through a disulfide bond to the ricin A chain with its N-glycosidase activity <cite>Lewis1986</cite>.  The ricin A chain  N-glycosidase cleaves a specific adenine from the pentose ribose in ribosomal RNA <cite>Endo1987</cite>. Finally, [[CBM29]] is a family with only two members, which have no appended catalytic modules; however, the function of these CBMs is to target the catalytic cellulosome machinery to substrate <cite>Freelove2001</cite>.
+Amino acid sequence-based classification of a CBM family may lead to the incorporation of other non-catalytic-associated CBMs within a given family. Some examples of families containing CBMs without appended catalytic modules include those with lectins (such as tachycitin ([[CBM14]]), wheat germ agglutinin ([[CBM18]]), fucolectin ([[CBM47]]), and malectin ([[CBM57]])), and those with periplasmic solute binding proteins ([[CBM32]]). Interestingly, the lectin ricin B chain ([[CBM13]]), while not on the same polypeptide chain, is covalently linked through a disulfide bond to the ricin A chain with its N-glycosidase activity <cite>Lewis1986</cite>.  The ricin A chain  N-glycosidase cleaves a specific adenine from the pentose ribose in ribosomal RNA <cite>Endo1987</cite>. Finally, [[CBM29]] is a family with only two members, which have no appended catalytic modules; however, the function of these CBMs is to target the catalytic cellulosome machinery to substrate <cite>Freelove2001</cite>.
 ==Studying CBM-ligand Interactions==

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 === Blurred Lines: CBMs, Lectins and Outliers ===
-While CBMs are generally considered to be discreet entities within a polypeptide chain, there are some exceptions. The glycogen-degrading pneumococcal virulence factor SpuA has its active site extended by one of two tandem [[CBM41]]s <cite>Lammerts2011</cite> and the glucan starch phosphatase Starch Excess4 has its active site extended by a [[CBM48]] <cite>Meekins2014</cite>. Thus, the full biological contribution to carbohydrate-binding within the polypeptide is contributed by a multivalent interaction as an extension of the catalytic module's carbohydrate-binding properties. The PA14 domain is found in bacterial toxins, enzymes, adhesins and signaling molecules <cite>Rigden2004</cite>.   It has been described as appended to the polypeptide sequence of some glycoside hydrolase enzymes (for example some [[GH31]]s) and the crystal structure of a [[GH31]] reveals the PA14 domain is closely associated with the catalytic module, on the side of the substrate-binding cleft, potentially facilitating the binding of longer oligosaccharides <cite>Larsbrink2011</cite>. It has also been described as a domain integrated into the core of some [[GH3]] glycoside hydrolase modules. In one example,  the [[GH3]] integrated PA14 domain demonstrates carbohydrate-binding function and acts to block the active site cleft, thus conferring substrate specificity for disaccharide substrates <cite>Yoshida2010</cite>. Similarly, in a [[GH2]] mannosidase, the PA14 domain determines exo- rather than endo-activity for the catalytic module <cite>Tailford2007</cite>. Evidently, more research needs to go into the structure and function of these domains as they are found in a wide variety of polypeptide sequences and the functions of the PA14 domains may be diverse.    They have not yet been classified into the CAZy classification system, though they are mentioned here as the domains have been referred to as CBMs in the literature <cite>Taylor2014</cite>.
+While CBMs are generally considered to be discrete entities within a polypeptide chain, there are some exceptions. The glycogen-degrading pneumococcal virulence factor SpuA has its active site extended by one of two tandem [[CBM41]]s <cite>Lammerts2011</cite> and the glucan starch phosphatase Starch Excess4 has its active site extended by a [[CBM48]] <cite>Meekins2014</cite>. Thus, the full biological contribution to carbohydrate-binding within the polypeptide is contributed by a multivalent interaction as an extension of the catalytic module's carbohydrate-binding properties. The PA14 domain is found in bacterial toxins, enzymes, adhesins and signaling molecules <cite>Rigden2004</cite>.   It has been described as appended to the polypeptide sequence of some glycoside hydrolase enzymes (for example some [[GH31]]s) and the crystal structure of a [[GH31]] reveals the PA14 domain is closely associated with the catalytic module, on the side of the substrate-binding cleft, potentially facilitating the binding of longer oligosaccharides <cite>Larsbrink2011</cite>. It has also been described as a domain integrated into the core of some [[GH3]] glycoside hydrolase modules. In one example,  the [[GH3]] integrated PA14 domain demonstrates carbohydrate-binding function and acts to block the active site cleft, thus conferring substrate specificity for disaccharide substrates <cite>Yoshida2010</cite>. Similarly, in a [[GH2]] mannosidase, the PA14 domain determines exo- rather than endo-activity for the catalytic module <cite>Tailford2007</cite>. Evidently, more research needs to go into the structure and function of these domains as they are found in a wide variety of polypeptide sequences and the functions of the PA14 domains may be diverse.    They have not yet been classified into the CAZy classification system, though they are mentioned here as the domains have been referred to as CBMs in the literature <cite>Taylor2014</cite>.
 Unrelated sugar-binding proteins have converged on similar biochemical mechanisms of saccharide recognition <cite>Taylor2014</cite>. The direct interaction of Ca<sup>2+</sup> ions with saccharides in sugar binding sites was first described in C-type animal lectins <cite>Weis1992</cite>, named thusly because of their sugar-binding requirement for Ca<sup>2+</sup>.  Other sugar-binding proteins that also require Ca<sup>2+</sup> for binding, include yeast flocculation proteins <cite>Veelders2010</cite> and other yeast adhesins <cite>Maestre-Reyna2012, Ielasi2012</cite>, and two CBM families, [[CBM36]] and [[CBM60]] <cite>Montanier2010</cite>.

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 Unrelated sugar-binding proteins have converged on similar biochemical mechanisms of saccharide recognition <cite>Taylor2014</cite>. The direct interaction of Ca<sup>2+</sup> ions with saccharides in sugar binding sites was first described in C-type animal lectins <cite>Weis1992</cite>, named thusly because of their sugar-binding requirement for Ca<sup>2+</sup>.  Other sugar-binding proteins that also require Ca<sup>2+</sup> for binding, include yeast flocculation proteins <cite>Veelders2010</cite> and other yeast adhesins <cite>Maestre-Reyna2012, Ielasi2012</cite>, and two CBM families, [[CBM36]] and [[CBM60]] <cite>Montanier2010</cite>.
-Several lectins <cite>SharonLis2004 SharonLis2007</cite> are classified as CBMs in the [http://www.cazy.org/Carbohydrate-Binding-Modules.html Carbohydrate Active enZyme database] as they share amino acid sequence similarity, exhibit similar folds and display similar carbohydrate binding properties. 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 <cite> Shallus2008 Galli2011</cite>. CBMs may also share properties with lectins that are not (yet) incorporated in the [http://www.cazy.org/Carbohydrate-Binding-Modules.html Carbohydrate Active enZyme database]. For example, the fucose-specific ''Anquila anguila'' lectin AAA was described as similar to Type C CBMs found in family [[CBM6]] and [[CBM32]] <cite>Boraston20032</cite> and is now classified as a [[CBM47]]<cite>Boraston2006</cite>. 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. A brief historical overview of the discovery and characterization of lectins is available <cite>SharonLis2004</cite> as is a review describing the convergent and divergent mechanisms of sugar recognition across the kingdoms of life <cite>Taylor2014</cite>.
+Several lectins <cite>SharonLis2004 SharonLis2007</cite> are classified as CBMs in the [http://www.cazy.org/Carbohydrate-Binding-Modules.html Carbohydrate Active enZyme database] as they share amino acid sequence similarity, exhibit similar folds and display similar carbohydrate binding properties. 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 <cite> Shallus2008 Galli2011</cite>. CBMs may also share properties with lectins that are not (yet) incorporated in the [http://www.cazy.org/Carbohydrate-Binding-Modules.html Carbohydrate Active enZyme database]. For example, the fucose-specific ''Anquila anguila'' lectin AAA was described as similar to Type C CBMs found in family [[CBM6]] and [[CBM32]] <cite>Boraston20032</cite> and is now classified as a [[CBM47]] <cite>Boraston2006</cite>. 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. A brief historical overview of the discovery and characterization of lectins is available <cite>SharonLis2004</cite> as is a review describing the convergent and divergent mechanisms of sugar recognition across the kingdoms of life <cite>Taylor2014</cite>.
 The biological reaction of agglutination is when particles that are suspended in a liquid collect into clumps, such as that occuring as a serologic response to a specific antibody. The most prominent feature that is genarally considered to separate CBMs from lectins is the involvement of lectins in agglutination of sugar-containing molecules or glycoconjugates.  Lectins exploit multivalency, often forming quaternary structures as homodimers, trimers or tetramers with several binding sites which then agglutinate the target glycocongugate <cite>SharonLis2004 SharonLis2007</cite>. Few studies have been done on the agglutinating effects of CBMs or CBM tandems; however, a [[CBM26]]/[[CBM25]] pair from ''Bacillus halodurans'' is described as strongly agglutinating on soluble amylopectin (and pullulan), suggesting multivalent binding of the individual CBMs to sites on separate glucan chains <cite>Boraston2006</cite>. CBMs individually are not known to be directly involved in the formation of quaternary structures and are not known to have agglutinating properties - in common with sugar-recognition modules of all glycan-binding proteins, including lectins <cite>Taylor2014</cite>. Other examples of CBMs participating in quaternary structures but not directly implicated in quaternary structure formation are found in cellulosome complexes <cite>Freelove2001 Poole1992 Morag1995</cite> and in some secreted pathogenic bacterial enzymes complexes <cite>Adams2008 Ficko2009</cite> where complex formation is mediated through specific cohesin-dockerin module interactions.

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 === Types ===
 [[Image:TypeAsurface.png|thumb|right|400px|'''Figure 3. CBM Types.''' (A) Schematic of different CBM Types binding with different regions of a polysaccharide substrate. (B) Type A [[CBM2]]b from ''Pyrococcus furiosis'' [[GH18]] chitinase(PDB ID [{{PDBlink}}2CRW 2CRW]) <cite>Nakamura2008</cite>. Aromatic side chains of Type A CBMs form the planar binding surface.]]
-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 [[#Functional Roles of CBMs|target]]. The classification of CBM Types is as follows <cite>Gilbert2013, Armenta2017, Boraston2004</cite>:
+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 saccharide macromolecule the enzyme will [[#Functional Roles of CBMs|target]]. The classification of CBM Types is as follows <cite>Gilbert2013, Armenta2017, Boraston2004</cite>:
-* 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 planar polycrystalline chitin/cellulose surface (Figure 3B). Type A CBMs are unique and differ significantly from Type B or C.
+* Type A: bind to crystalline surfaces of the polysaccharides 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 planar 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). Type B are the most abundant form of CBMs reported to date. Type B binding sites appear as extended grooves or clefts comprised of binding subsites to generally accommodate longer sugar chains with four or more monosaccharide units (see Figure 2A for an example). There are some examples of CBMs, in families [[CBM6]], [[CBM13]], [[CBM20]], [[CBM36]] and [[CBM60]], that contain  two subsites.
 * 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]], [[CBM32]], [[CBM47]], [[CBM66]], [[CBM67]]).  Families containing Type C CBMs are considered 'lectin-like' and may include lectins and CBMs with no appended catalytic modules as members.
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 === CBMs and Multivalency ===
-Multivalency is the collective strength of several interactions with a given ligand. Because CBM-carbohydrate interactions are relatively weak, some carbohydrate-active enzymes, mainly glycoside hydrolases, have developed ways to increase their interaction with substrate via a multivalent effect. Individually, some CBMs may contain multiple binding sites to form a multivalent interaction with their target ligand, although this form of multivalency is quite rare (for example [[CBM6]], [[CBM13]] and [[CBM20]]). More commonly, glycoside hydrolases may contain more than one CBM within their modular architecture, either arranged in tandem or at opposing N and C terminal ends of the protein sequence, or both. These CBMs may target the same carbohydrate ligand, different regions within the same ligand, or different ligands in a complex saccharide amalgam. A multivalent interaction enhances the overall affinity of an enzyme for its substrate but more importantly, tandem CBMs will cooperatively target the enzyme towards specific regions based on the orientation and position of binding sites with respect to one another.
+Multivalency is the collective strength of several interactions with a given ligand. Because CBM-carbohydrate interactions are relatively weak, some carbohydrate-active enzymes, mainly glycoside hydrolases, have developed ways to increase their interaction with substrate via a multivalent effect. Individually, some CBMs may contain multiple binding sites to form a multivalent interaction with their target ligand, although this form of multivalency is quite rare (for example [[CBM6]], [[CBM13]] and [[CBM20]]). More commonly, glycoside hydrolases may contain more than one CBM within their modular architecture, either arranged in tandem or at opposing N and C terminal ends of the protein sequence, or both. These CBMs may target the same carbohydrate ligand, different regions within the same ligand, or different ligands in a complex saccharide amalgam. A multivalent interaction enhances the overall affinity of an enzyme for its substrate. Furthermore, tandem CBMs may cooperatively target the enzyme towards specific saccharide regions based on their ligand specificity and the orientation and position of the binding sites with respect to one another.
 === Blurred Lines: CBMs, Lectins and Outliers ===