Carbohydrate Binding Module Family 35 - Revision history

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

2021-12-18T21:15:56Z

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

Elizabeth Ficko-Blean: /* Structural features */

2018-03-06T16:11:03Z

Structural features

Elizabeth Ficko-Blean: /* Structural features */

2018-03-06T16:10:42Z

Structural features

Harry Gilbert: /* Functionalities */

2018-02-05T22:50:48Z

Functionalities

Harry Gilbert: /* Structural features */

2018-02-05T22:46:27Z

Structural features

Harry Gilbert: /* Structural features */

2018-02-05T22:46:07Z

Structural features

Harry Brumer: /* Family Firsts */

2018-01-31T19:01:09Z

Family Firsts

Harry Gilbert at 12:15, 31 January 2018

2018-01-31T12:15:57Z

Harry Gilbert at 12:14, 31 January 2018

2018-01-31T12:14:36Z

Harry Gilbert at 18:45, 28 January 2018

2018-01-28T18:45:06Z

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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]]: ^^^Harry Gilbert^^^
+* [[Author]]: [[User:Harry Gilbert|Harry Gilbert]]
-* [[Responsible Curator]]:  ^^^Wade Abbott^^^
+* [[Responsible Curator]]:  [[User:Wade Abbott|Wade Abbott]]
 ----

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 [[File:CBM35-glucan complex.png|thumb|300px|right|'''Figure 3.'''  An overlay of the ligand binding site of BcCBM35-1 ([{{PDBlink}}3WMN PDB ID 3WMN]; green) and BcCBM35-2 ([{{PDBlink}}5X7Q PDB ID 5X7Q]; cyan) in complex with &alpha;1,6- (green) and &alpha;1,4-glucans (cyan), respectively. The two ligand binding sites are in close but not identical positions in the two proteins. The ligand binding residues of the two CBMs comprise both conserved and distinct amino acids. Note the the binding sites do not contain a calcium and the lack of the arginine (present in the UA-CBM35) enables extension of the &alpha;1,6-glucan at O6.]]
-The structures of CBM35s with different specificities have been reported. All these proteins adopt a β-sandwich fold in which the two β-sheets contain four and five antiparallel β-strands, respectively. In one of the glucan binding CBM35s the concave surface of one of the &beta;-sheets; comprises the binding site for a circular tetrasaccharide (see below). In all the other CBM35s, however, the ligand binding site is located in the "Variable Loop Site" <cite>Abbott2014</cite> that plays a role in connecting the two β-sheets  (Figure 1). In the UA-CBM35s a calcium is located in the ligand binding site that interacts via a solvent molecule with the carboxylate (Figure 1 <cite>Montanier2009</cite>). The Asn ligands for the metal ion also hydrogen bond with the sugar and, by making interactions only with an equatorial O4 select for GlcA over GalA. A conserved arginine makes bidentate polar interactions with the carboxylate and a tryptophan forms parallel hydrophobic interactions with the pyranose ring. Subtle changes in the polar residues in the ligand binding site of a pectate lyase CBM35 confers specificity for only the unsaturated uronic acid. The role of these polar and hydrophobic residues in glycan binding was confirmed by mutagenesis. The pocket topology of the ligand binding site in which only O1 is solvent exposed explains why these CBMs are exo-binding, recognising the non-reducing terminal sugars and are thus classified as [[Carbohydrate-binding_modules#Types|type C]] modules.
+The structures of CBM35s with different specificities have been reported. All these proteins adopt a β-sandwich fold in which the two β-sheets contain four and five antiparallel β-strands, respectively. In one of the glucan binding CBM35s the concave surface of one of the &beta;-sheets; comprises the binding site for a circular tetrasaccharide (see below). In all the other CBM35s, however, the ligand binding site is located in the "Variable Loop Site" <cite>Abbott2014</cite> that plays a role in connecting the two β-sheets  (Figure 1). In the UA-CBM35s a calcium is located in the ligand binding site that interacts via a solvent molecule with the carboxylate (Figure 1 <cite>Montanier2009</cite>). The Asn ligands for the metal ion also hydrogen bond with the sugar and, by making interactions only with an equatorial O4 select for GlcA over GalA. A conserved arginine makes bidentate polar interactions with the carboxylate and a tryptophan forms parallel hydrophobic interactions with the pyranose ring. Subtle changes in the polar residues in the ligand binding site of a pectate lyase CBM35 confers specificity for only the unsaturated uronic acid. The role of these polar and hydrophobic residues in glycan binding was confirmed by mutagenesis. The pocket topology of the ligand binding site in which only O1 is solvent exposed explains why these CBMs are exo-binding, recognising the non-reducing terminal sugars and are thus classified as [[Carbohydrate-binding_modules#Types|type C]] CBMs.
 The ligand binding site of the CBM35 that binds to &beta;-mannan (Man-CBM35) adopts a cleft topology and, reflecting its endo-binding mode is designated a a type B module <cite>Tunnicliffe2005</cite>. In contrast to the majority of CBMs, which are generally rigid proteins, Man-CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s.

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 [[File:CBM35-glucan complex.png|thumb|300px|right|'''Figure 3.'''  An overlay of the ligand binding site of BcCBM35-1 ([{{PDBlink}}3WMN PDB ID 3WMN]; green) and BcCBM35-2 ([{{PDBlink}}5X7Q PDB ID 5X7Q]; cyan) in complex with &alpha;1,6- (green) and &alpha;1,4-glucans (cyan), respectively. The two ligand binding sites are in close but not identical positions in the two proteins. The ligand binding residues of the two CBMs comprise both conserved and distinct amino acids. Note the the binding sites do not contain a calcium and the lack of the arginine (present in the UA-CBM35) enables extension of the &alpha;1,6-glucan at O6.]]
-The structures of CBM35s with different specificities have been reported. All these proteins adopt a β-sandwich fold in which the two β-sheets contain four and five antiparallel β-strands, respectively. In one of the glucan binding CBM35s the concave surface of one of the &beta;-sheets; comprises the binding site for a circular tetrasaccharide (see below). In all the other CBM35s, however, the ligand binding site is located in the "Variable Loop Site" <cite>Abbott2014</cite> that plays a role in connecting the two β-sheets  (Figure 1). In the UA-CBM35s a calcium is located in the ligand binding site that interacts via a solvent molecule with the carboxylate (Figure 1 <cite>Montanier2009</cite>). The Asn ligands for the metal ion also hydrogen bond with the sugar and, by making interactions only with an equatorial O4 select for GlcA over GalA. A conserved arginine makes bidentate polar interactions with the carboxylate and a tryptophan forms parallel hydrophobic interactions with the pyranose ring. Subtle changes in the polar residues in the ligand binding site of a pectate lyase CBM35 confers specificity for only the unsaturated uronic acid. The role of these polar and hydrophobic residues in glycan binding was confirmed by mutagenesis. The pocket topology of the ligand binding site in which only O1 is solvent exposed explains why these CBMs are exo-binding, recognising the non-reducing terminal sugars and are thus classified as type C modules.
+The structures of CBM35s with different specificities have been reported. All these proteins adopt a β-sandwich fold in which the two β-sheets contain four and five antiparallel β-strands, respectively. In one of the glucan binding CBM35s the concave surface of one of the &beta;-sheets; comprises the binding site for a circular tetrasaccharide (see below). In all the other CBM35s, however, the ligand binding site is located in the "Variable Loop Site" <cite>Abbott2014</cite> that plays a role in connecting the two β-sheets  (Figure 1). In the UA-CBM35s a calcium is located in the ligand binding site that interacts via a solvent molecule with the carboxylate (Figure 1 <cite>Montanier2009</cite>). The Asn ligands for the metal ion also hydrogen bond with the sugar and, by making interactions only with an equatorial O4 select for GlcA over GalA. A conserved arginine makes bidentate polar interactions with the carboxylate and a tryptophan forms parallel hydrophobic interactions with the pyranose ring. Subtle changes in the polar residues in the ligand binding site of a pectate lyase CBM35 confers specificity for only the unsaturated uronic acid. The role of these polar and hydrophobic residues in glycan binding was confirmed by mutagenesis. The pocket topology of the ligand binding site in which only O1 is solvent exposed explains why these CBMs are exo-binding, recognising the non-reducing terminal sugars and are thus classified as [[Carbohydrate-binding_modules#Types|type C]] modules.
 The ligand binding site of the CBM35 that binds to &beta;-mannan (Man-CBM35) adopts a cleft topology and, reflecting its endo-binding mode is designated a a type B module <cite>Tunnicliffe2005</cite>. In contrast to the majority of CBMs, which are generally rigid proteins, Man-CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s.

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 == Functionalities ==
-Although CBM35 constitutes a discrete protein family, phylogenetic analysis showed that it displays a distant relationship with family 6 CBMs <cite>Bolam2004</cite>. Based on sequence similarities Family 35 CBMs partitions into four major clades (I-IV). Functional predictions for each subfamily are based upon the distributions of CBM35s with known specificities, including carbohydrates with galacto (I), manno (II), gluco (III), and uronate (IV) configurations <cite>Correia2010</cite>. The analysis reveals some enzymes that contain multiple copies of CBM35s that cluster into different subfamilies, illustrated by BcCB35-1 and BcCBM35-2, described above, or are located in the same subfamily suggesting conserved ligand recognition leading to enhanced affinity through avidity effects. A more detailed phylogenetic analysis of CBM35 identified five molecular regions that contribute to specificity and affinity <cite>Correia2010</cite>. The functional significance of CBM35s have been discussed in the literature. It has been proposed that the recognition of Δ4,5-GalA  by CBM35s that are components of plant cell wall (PCW) degrading enzymes, indicates that these CBMs play a role in targeting their cognate CAZymes to regions of the PCW accessible to biological degradation. Pectins are arguably the most accessible region of the plant cell wall and indeed cloak other polysaccharides, such as xyloglucan, occluding them from enzyme attack  <cite>Marcus2008</cite>. Thus, by targeting enzymes to regions rich in Δ4,5-GalA, the UA-CBM35s are directing their cognate catalytic modules to areas of the PCW that are particularly accessible to further biological degradation. This hypothesis was supported by extensive binding of the UA-CBM35 to the pectin rich primary cell walls of plants only after pectate lyase treatment <cite>Montanier2009</cite>. It should be emphasized, however, that a group of three UA-CBM35s form cassettes with cellulose binding CBM2s (these CBM2-UA-CBM35 cassettes are identical in amino acid sequence even at the nucleotide level <cite>Kellett1990,Ferreira1993</cite>), and are appended to three xylan degrading enzymes (esterase, arabinofuranosidase and endo-xylanase). These UA-CBM35s also display significant affinity for GlcA, a side chain present in several xylans <cite>Pena2007</cite>. Thus, this plasticity in uronic acid recognition provides the enzyme with a flexible targeting strategy that is tailored to the plant biomass presented to the bacterium. It was argued that this cohort of CBM35s directed their enzymes to regions of cell walls that are being actively degraded but, as xylan structures are revealed, the enzyme is shuttled onto the hemicellulosic polysaccharide affording the hydrolases access to its target substrate <cite>Montanier2009</cite>. Indeed appending the UA-CBM35 to a xylanase enhanced its activity against plant cell walls in muro <cite> Urbanowicz2012</cite>. It was also proposed shown that a UA-CBM35, appended to an exo-β-d-glucosaminidase from the soil bacterium ''Amycolatopsis orientalis'' interacts with the cell wall of the organism, which are known to contain GlcA. Immunofluorescence microscopy showed that the UA-CBM functions as an adhesion molecule tethering the exo-β-D-glucosaminidase to the bacterial cell surface <cite>Montanier2009</cite>. The &alpha;-glucan binding CBMs in glucantransferases appear to contribute to substrate binding in concert with the catalytic module. For example BcCBM35-1 appears to contribute the -8 subsite of the enzyme <cite>Suzuki2014</cite>. This hypothesis was supported by mutagenensis of the two sugar binding sites of BcCBM35-1, while led to a substantial increase in the proportion of large cycloisomaltooligosaccharides with a degree of polymerization <9. In addition to influencing enzyme activity, UA-CBM35s have also been used to explore the nature of GlcA in PCW, as these modules bind to GlcA but not 4-Me-O-GlcA <cite>Urbanowicz2012</cite>
+Although CBM35 constitutes a discrete protein family, phylogenetic analysis showed that it displays a distant relationship with family 6 CBMs <cite>Bolam2004</cite>. Based on sequence similarities Family 35 CBMs partitions into four major clades (I-IV). Functional predictions for each subfamily are based upon the distributions of CBM35s with known specificities, including carbohydrates with galacto (I), manno (II), gluco (III), and uronate (IV) configurations <cite>Correia2010</cite>. The analysis reveals some enzymes that contain multiple copies of CBM35s that cluster into different subfamilies, illustrated by BcCB35-1 and BcCBM35-2, described above, or are located in the same subfamily suggesting conserved ligand recognition leading to enhanced affinity through avidity effects. A more detailed phylogenetic analysis of CBM35 identified five molecular regions that contribute to specificity and affinity <cite>Correia2010</cite>.
 == Family Firsts ==

@@ Line 25: / Line 25: @@
 [[File:UA-CBM35complex.png|thumb|300px|right|'''Figure 2.'''  The structure of UA-CBM35 in complex with glucuronic acid showing the role played by the two asparagines in orientating a calcium to make polar contacts with the uronic acid. The apolar interactions between the pyranose ring and a conserved tryptophan, and the bidentate polar interactions between the carboxylate and an arginine are key components of ligand recognition.]]
-[[
+[[File:CBM35-glucan complex.png|thumb|300px|right|'''Figure 3.'''  An overlay of the ligand binding site of BcCBM35-1 ([{{PDBlink}}3WMN PDB ID 3WMN]; green) and BcCBM35-2 ([{{PDBlink}}5X7Q PDB ID 5X7Q]; cyan) in complex with &alpha;1,6- (green) and &alpha;1,4-glucans (cyan), respectively. The two ligand binding sites are in close but not identical positions in the two proteins. The ligand binding residues of the two CBMs comprise both conserved and distinct amino acids. Note the the binding sites do not contain a calcium and the lack of the arginine (present in the UA-CBM35) enables extension of the &alpha;1,6-glucan at O6.]]
 The structures of CBM35s with different specificities have been reported. All these proteins adopt a β-sandwich fold in which the two β-sheets contain four and five antiparallel β-strands, respectively. In one of the glucan binding CBM35s the concave surface of one of the &beta;-sheets; comprises the binding site for a circular tetrasaccharide (see below). In all the other CBM35s, however, the ligand binding site is located in the "Variable Loop Site" <cite>Abbott2014</cite> that plays a role in connecting the two β-sheets  (Figure 1). In the UA-CBM35s a calcium is located in the ligand binding site that interacts via a solvent molecule with the carboxylate (Figure 1 <cite>Montanier2009</cite>). The Asn ligands for the metal ion also hydrogen bond with the sugar and, by making interactions only with an equatorial O4 select for GlcA over GalA. A conserved arginine makes bidentate polar interactions with the carboxylate and a tryptophan forms parallel hydrophobic interactions with the pyranose ring. Subtle changes in the polar residues in the ligand binding site of a pectate lyase CBM35 confers specificity for only the unsaturated uronic acid. The role of these polar and hydrophobic residues in glycan binding was confirmed by mutagenesis. The pocket topology of the ligand binding site in which only O1 is solvent exposed explains why these CBMs are exo-binding, recognising the non-reducing terminal sugars and are thus classified as type C modules.

@@ Line 31: / Line 31: @@
 == Family Firsts ==
-;First Identified
+;First Identified: The Man-CBM35 and a UA-CBM35 from a ''C. japonicus'' GH5 mannanase and GH62 arabinofuranosidase, respectively, were the first members of this family to be identified and characterized <cite>Bolam2004</cite>.
 The Man-CBM35 and a UA-CBM35 from a ''C. japonicus'' GH5 mannanase and GH62 arabinofuranosidase, respectively, were the first members of this family to be identified and characterized <cite>Bolam2004</cite>.
 == References ==
 <biblio>

@@ Line 28: / Line 28: @@
 The structures of CBM35s with different specificities have been reported. All these proteins adopt a β-sandwich fold in which the two β-sheets contain four and five antiparallel β-strands, respectively. In the majority of CBM35s the ligand binding site is located in the loops in the Variable Loop Site <cite>Abbott2014</cite> that plays a role in connecting the two β-sheets  (Figure 1). In the UA-CBM35s a calcium is located in the ligand binding site that interacts via a solvent molecule with the carboxylate (Figure 1 <cite>Montanier2009</cite>). The Asn ligands for the metal ion also hydrogen bond with the sugar and, by making interactions only with an equatorial O4 select for GlcA over GalA. A conserved arginine makes bidentate polar interactions with the carboxylate and a tryptophan forms parallel hydrophobic interactions with the pyranose ring. Subtle changes in the polar residues in the ligand binding site of a pectate lyase CBM35 confers specificity for only the unsaturated uronic acid. The role of these polar and hydrophobic residues in glycan binding was confirmed by mutagenesis. The pocket topology of the ligand binding site in which only O1 is solvent exposed explains why these CBMs are exo-binding, recognising the non-reducing terminal sugars and are thus classified as type C modules. The ligand binding site of the CBM that binds to &beta;-mannan (Man-CBM35) adopts a cleft topology and, reflecting its endo-binding mode is designated a a type B module <cite>Tunnicliffe2005</cite>. In contrast to the majority of CBMs, which are generally rigid proteins, Man-CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s. There are also a very large number of structures of CBM35s derived from glucantransferases <cite>Suzuki2014,Fujimoto2017,Light2017</cite>, with several of these enzymes containing multiple copies of these modules. For example a CBM35 module (BcCBM35-1) is located in the loop connecting the &beta;7 strand and &alpha;-helix of the TIM barrel GH66 catalytic module of a Bacillus circulans cycloisomaltooligosaccharide glucanotransferase, while a second CBM35 (BcCBM35-2) in this enzyme is a discrete module <cite>Suzuki2014</cite>. BcCBM35-1 and BcCBM35-2 bind to &alpha;1,6- and &alpha;1,4-glucans, respectively, in the same region described above for the UA-CBM35s. In these CBMs there is no calcium in the ligand binding site, however, the crucial stacking tryptophan residue is conserved. The ligand binding site of BcCBM35-1 has a pocket-like topology, similar to UA-CBM35s. The pocket, however, lacks the conserved arginine in UA-CBMs that makes polar interactions with the carboxylate of GlcA ligand (Figure 2). The lack of this bulky amino acid creates the necessary space for O6 to be solvent exposed, explaining why the CBM can interact with internal sugars in the highly curved structure of &alpha;1,6-glucans. The binding site for &alpha;1,4-glucans in CBM35-2 is displaced by 11 &angstrongs; from the binding pocket in the other CBM35s. In addition to making polar contacts with ligand through residues that are conserved in the other CBM35s, BcCBM35-2 also contains a  surface tryptophan and two asparagine residues, absent in other family members, that make strong hydrogen bonds and apolar interactions with the &alpha;1,4-glucan (Figure 3). In the CBM35 of the Listeria monocytogenes glucantransferase CAFÉ, an alternating &alpha;1,3-&alpha;1,6- circular tetrasaccharide binds on the concave surface adopted by one of the &beta;-sheets <cite>Light2017</cite> ([{{PDBlink}}5i0d PDB ID 5i0d]).
 == Functionalities ==
-Although CBM35 constitutes a discrete protein family, phylogenetic analysis showed that it displays a distant relationship with family 6 CBMs <cite>Bolam2004</cite>. Based on sequence similarities Family 35 CBMs partitions into four major clades (I-IV). Functional predictions for each subfamily are based upon the distributions of CBM35s with known specificities, including carbohydrates with galacto (I), manno (II), gluco (III), and uronate (IV) configurations <cite>Correia2010</cite>. The analysis reveals some enzymes that contain multiple copies of CBM35s that cluster into different subfamilies, illustrated by BcCB35-1 and BcCBM35-2, described above, or are located in the same subfamily suggesting conserved ligand recognition leading to enhanced affinity through avidity effects. A more detailed phylogenetic analysis of CBM35 identified five molecular regions that contribute to specificity and affinity <cite>Correia2010</cite>. The functional significance of CBM35s have been discussed in the literature. It has been proposed that the recognition of Δ4,5-GalA  by CBM35s that are components of plant cell wall (PCW) degrading enzymes, indicates that these CBMs play a role in targeting their cognate CAZymes to regions of the PCW accessible to biological degradation. Pectins are arguably the most accessible region of the plant cell wall and indeed cloak other polysaccharides, such as xyloglucan, occluding them from enzyme attack  <cite>Marcus2008</cite>. Thus, by targeting enzymes to regions rich in Δ4,5-GalA, the UA-CBM35s are directing their cognate catalytic modules to areas of the PCW that are particularly accessible to further biological degradation. This hypothesis was supported by extensive binding of the UA-CBM35 to the pectin rich primary cell walls of plants only after pectate lyase treatment <cite>Montanier2009</cite>. It should be emphasized, however, that a group of three UA-CBM35s form cassettes with cellulose binding CBM2s (these CBM2-UA-CBM35 cassettes are identical in amino acid sequence even at the nucleotide level <cite>Kellett1990,Ferreira1993</cite>), and are appended to three xylan degrading enzymes (esterase, arabinofuranosidase and endo-xylanase). These UA-CBM35s also display significant affinity for GlcA, a side chain present in several xylans <cite>Pena2007</cite>. Thus, this plasticity in uronic acid recognition provides the enzyme with a flexible targeting strategy that is tailored to the plant biomass presented to the bacterium. It was argued that this cohort of CBM35s directed their enzymes to regions of cell walls that are being actively degraded but, as xylan structures are revealed, the enzyme is shuttled onto the hemicellulosic polysaccharide affording the hydrolases access to its target substrate <cite>Montanier2009</cite>. Indeed appending the UA-CBM35 to a xylanase enhanced its activity against plant cell walls in muro <cite> Urbanowicz2012</cite>. It was also proposed shown that a UA-CBM35, appended to an exo-β-d-glucosaminidase from the soil bacterium Amycolatopsis orientalis interacts with the cell wall of the organism, which are known to contain GlcA. Immunofluorescence microscopy showed that the UA-CBM functions as an adhesion molecule tethering the exo-β-d-glucosaminidase to the bacterial cell surface <cite>Montanier2009</cite>. The &alpha-glucan; binding CBMs in glucantransferases appear to contribute to substrate binding in concert with the catalytic module. For example BcCBM35-1 appears to contribute the -8 subsite of the enzyme <cite>Suzuki2014</cite>. This hypothesis was supported by mutagenensis of the two sugar binding sites of BcCBM35-1, while led to a substantial increase in the proportion of large cycloisomaltooligosaccharides with a degree of polymerization <9. In addition to influencing enzyme activity, UA-CBM35s have also been used to explore the nature of GlcA in PCW, as these modules bind to GlcA but not 4-Me-O-GlcA <cite>Urbanowicz2012</cite>
+Although CBM35 constitutes a discrete protein family, phylogenetic analysis showed that it displays a distant relationship with family 6 CBMs <cite>Bolam2004</cite>. Based on sequence similarities Family 35 CBMs partitions into four major clades (I-IV). Functional predictions for each subfamily are based upon the distributions of CBM35s with known specificities, including carbohydrates with galacto (I), manno (II), gluco (III), and uronate (IV) configurations <cite>Correia2010</cite>. The analysis reveals some enzymes that contain multiple copies of CBM35s that cluster into different subfamilies, illustrated by BcCB35-1 and BcCBM35-2, described above, or are located in the same subfamily suggesting conserved ligand recognition leading to enhanced affinity through avidity effects. A more detailed phylogenetic analysis of CBM35 identified five molecular regions that contribute to specificity and affinity <cite>Correia2010</cite>. The functional significance of CBM35s have been discussed in the literature. It has been proposed that the recognition of Δ4,5-GalA  by CBM35s that are components of plant cell wall (PCW) degrading enzymes, indicates that these CBMs play a role in targeting their cognate CAZymes to regions of the PCW accessible to biological degradation. Pectins are arguably the most accessible region of the plant cell wall and indeed cloak other polysaccharides, such as xyloglucan, occluding them from enzyme attack  <cite>Marcus2008</cite>. Thus, by targeting enzymes to regions rich in Δ4,5-GalA, the UA-CBM35s are directing their cognate catalytic modules to areas of the PCW that are particularly accessible to further biological degradation. This hypothesis was supported by extensive binding of the UA-CBM35 to the pectin rich primary cell walls of plants only after pectate lyase treatment <cite>Montanier2009</cite>. It should be emphasized, however, that a group of three UA-CBM35s form cassettes with cellulose binding CBM2s (these CBM2-UA-CBM35 cassettes are identical in amino acid sequence even at the nucleotide level <cite>Kellett1990,Ferreira1993</cite>), and are appended to three xylan degrading enzymes (esterase, arabinofuranosidase and endo-xylanase). These UA-CBM35s also display significant affinity for GlcA, a side chain present in several xylans <cite>Pena2007</cite>. Thus, this plasticity in uronic acid recognition provides the enzyme with a flexible targeting strategy that is tailored to the plant biomass presented to the bacterium. It was argued that this cohort of CBM35s directed their enzymes to regions of cell walls that are being actively degraded but, as xylan structures are revealed, the enzyme is shuttled onto the hemicellulosic polysaccharide affording the hydrolases access to its target substrate <cite>Montanier2009</cite>. Indeed appending the UA-CBM35 to a xylanase enhanced its activity against plant cell walls in muro <cite> Urbanowicz2012</cite>. It was also proposed shown that a UA-CBM35, appended to an exo-β-d-glucosaminidase from the soil bacterium Amycolatopsis orientalis interacts with the cell wall of the organism, which are known to contain GlcA. Immunofluorescence microscopy showed that the UA-CBM functions as an adhesion molecule tethering the exo-β-D-glucosaminidase to the bacterial cell surface <cite>Montanier2009</cite>. The &alpha;-glucan binding CBMs in glucantransferases appear to contribute to substrate binding in concert with the catalytic module. For example BcCBM35-1 appears to contribute the -8 subsite of the enzyme <cite>Suzuki2014</cite>. This hypothesis was supported by mutagenensis of the two sugar binding sites of BcCBM35-1, while led to a substantial increase in the proportion of large cycloisomaltooligosaccharides with a degree of polymerization <9. In addition to influencing enzyme activity, UA-CBM35s have also been used to explore the nature of GlcA in PCW, as these modules bind to GlcA but not 4-Me-O-GlcA <cite>Urbanowicz2012</cite>
 == Family Firsts ==