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Difference between revisions of "Carbohydrate Binding Module Family 67"

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== Structural Features ==
 
== Structural Features ==
SaCBM67 is ʟ-rhamnose binding module. SaCBM67 is thus a type C CBM. SaRha78A (pdb: 3W5N) forms a multidomain structure comprised of six distinct domains, one α-domain and five β-domains (domains N, D, E, F, A (catalytic module designated SaRha78<sub>CM</sub>), and C. Domain D designated SaCBM67 (Pro133-Pro297). SaCBM67 displays weak structural homology with a CBM32, CBM35, CBM36, and CBM60, which recognize their different ligands through either an exo- (CBM32 and CBM35) or endo-mode (CBM36 and CBM60) of binding. These CBMs all comprise a β-jellyroll structure and contain a second calcium atom that is integral to ligand recognition, in addition to a structural calcium (which is absent in CBM67). A central feature of the ʟ-rhamnose binding site in SaCBM67 (see Fig. 3C of <cite>Fujimoto2013</cite>) is a calcium ion that makes coordinate bonds with O3 and O4 of the sugar. The calcium interacts with SaCBM67 through D179, N180, N228, P233, and a water-mediated contact with S230. The bound ʟ-rhamnose also makes direct hydrogen bonds with W203, N180, and D179 through O2, O3, and O4 atoms, respectively. The C-6 methyl group pointed toward a small hydrophobic pocket comprising W203, P233, P291 and W292. No direct interaction with C-6 methyl group well explained why SaCBM67 also bind to ʟ-mannose.
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SaCBM67 is a ʟ-rhamnose binding module. SaCBM67 is thus a type C CBM. SaRha78A (pdb: 3W5N) forms a multidomain structure comprised of six distinct domains, one α-domain and five β-domains (domains N, D, E, F, A (catalytic module designated SaRha78<sub>CM</sub>), and C. Domain D designated SaCBM67 (P133-P297). SaCBM67 displays weak structural homology with a CBM32, CBM35, CBM36, and CBM60, which recognize their different ligands through either an exo- (CBM32 and CBM35) or endo-mode (CBM36 and CBM60) of binding. These CBMs all comprise a β-jellyroll structure and contain a second calcium atom that is integral to ligand recognition, in addition to a structural calcium (which is absent in CBM67). A central feature of the ʟ-rhamnose binding site in SaCBM67 (see Fig. 3C of <cite>Fujimoto2013</cite>) is a calcium ion that makes coordinate bonds with O3 and O4 of the sugar. The calcium interacts with SaCBM67 through D179, N180, N228, P233, and a water-mediated contact with S230. The bound ʟ-rhamnose also makes direct hydrogen bonds with W203, N180, and D179 through O2, O3, and O4 atoms, respectively. The C-6 methyl group pointed toward a small hydrophobic pocket comprising W203, P233, P291 and W292. No direct interaction with C-6 methyl group well explained why SaCBM67 also bind to ʟ-mannose.
  
 
== Functionalities ==  
 
== Functionalities ==  
Although SaCBM67 showed 2 times higher affinity for ʟ-mannose than ʟ-rhamnose, SaCBM67 binds primarily to ʟ-rhamnose in biological systems because ʟ-mannose seldom exists in natural polysaccharides. Actually mutant enzymes deleted calcium-mediated and direct hydrogen bonds with ʟ-rhamnose caused a substantial reduction (∼50-fold) in activity against the ʟ-rhamnose-containing polysaccharide (the mutantions did not influence activity against aryl-rhamnosides) <cite>Fujimoto2013</cite>.
+
Although SaCBM67 showed 2 times higher affinity for ʟ-mannose than ʟ-rhamnose, it binds primarily to ʟ-rhamnose in biological systems because ʟ-mannose seldom exists in natural polysaccharides. Actually mutant enzymes deleted calcium-mediated and direct hydrogen bonds with ʟ-rhamnose caused a substantial reduction (∼50-fold) in activity against the ʟ-rhamnose-containing polysaccharide (the mutantions did not influence activity against aryl-rhamnosides) <cite>Fujimoto2013</cite>.
  
 
CBM67 members are distributed not only in many bacterial GH78 α-ʟ-rhamnosidases, but also in some Basidiomycete lectins, family 1 pectate lyases, peptidases, and proteins of unknown functions. Protein alignment of candidate members of CBM67 identified five subfamilies within the constructed phylogenetic tree. Although the calcium binding site is conserved in the CBM67, these CBM67 might show different sugar specificity because the ʟ-rhamnose binding residues in SaCBM67 are not retaining in the other CBM67 members. For example, the lectin from ''Pleurotus cornucopiae'' contains two CBM67-like sequences in tandem with sequence identities against SaCBM67 of 25 and 35% for the N- and C-terminal modules respectively, shows highest affinity against ''N''-acetyl-ᴅ-galactosamine.
 
CBM67 members are distributed not only in many bacterial GH78 α-ʟ-rhamnosidases, but also in some Basidiomycete lectins, family 1 pectate lyases, peptidases, and proteins of unknown functions. Protein alignment of candidate members of CBM67 identified five subfamilies within the constructed phylogenetic tree. Although the calcium binding site is conserved in the CBM67, these CBM67 might show different sugar specificity because the ʟ-rhamnose binding residues in SaCBM67 are not retaining in the other CBM67 members. For example, the lectin from ''Pleurotus cornucopiae'' contains two CBM67-like sequences in tandem with sequence identities against SaCBM67 of 25 and 35% for the N- and C-terminal modules respectively, shows highest affinity against ''N''-acetyl-ᴅ-galactosamine.

Revision as of 21:35, 10 June 2018

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CAZy DB link
http://www.cazy.org/CBM67.html

Ligand specificities

The sugar binding structure of a GH78 α-ʟ-rhamnosidase from Streptomyces avermitilis (SaRha78A) revealed a ʟ-rhamnose binding module CBM67 (SaCBM67) within the six-domain arrangement [1]. SaCBM67 bound ʟ-rhamnose and ʟ-mannose with a Ka of 7.2 × 103 M−1 and 3.6 × 103 M−1, and free energy of binding ΔG of −5.3 kcal/mol and −4.8 kcal/mol, respectively, but did not bind to ʟ-rhamnose in the presence of 5 mM EDTA [1]. Similarly the D179A and N180A mutants of SaCBM67, in which removed calcium-mediated and direct hydrogen bonds with ʟ-rhamnose, abolish ligand binding, confirming the importance of calcium in the binding of SaCBM67 to its ligand [1]. No binding to ʟ-galactose or ʟ-fucose was also observed [1].

Structural Features

SaCBM67 is a ʟ-rhamnose binding module. SaCBM67 is thus a type C CBM. SaRha78A (pdb: 3W5N) forms a multidomain structure comprised of six distinct domains, one α-domain and five β-domains (domains N, D, E, F, A (catalytic module designated SaRha78CM), and C. Domain D designated SaCBM67 (P133-P297). SaCBM67 displays weak structural homology with a CBM32, CBM35, CBM36, and CBM60, which recognize their different ligands through either an exo- (CBM32 and CBM35) or endo-mode (CBM36 and CBM60) of binding. These CBMs all comprise a β-jellyroll structure and contain a second calcium atom that is integral to ligand recognition, in addition to a structural calcium (which is absent in CBM67). A central feature of the ʟ-rhamnose binding site in SaCBM67 (see Fig. 3C of [1]) is a calcium ion that makes coordinate bonds with O3 and O4 of the sugar. The calcium interacts with SaCBM67 through D179, N180, N228, P233, and a water-mediated contact with S230. The bound ʟ-rhamnose also makes direct hydrogen bonds with W203, N180, and D179 through O2, O3, and O4 atoms, respectively. The C-6 methyl group pointed toward a small hydrophobic pocket comprising W203, P233, P291 and W292. No direct interaction with C-6 methyl group well explained why SaCBM67 also bind to ʟ-mannose.

Functionalities

Although SaCBM67 showed 2 times higher affinity for ʟ-mannose than ʟ-rhamnose, it binds primarily to ʟ-rhamnose in biological systems because ʟ-mannose seldom exists in natural polysaccharides. Actually mutant enzymes deleted calcium-mediated and direct hydrogen bonds with ʟ-rhamnose caused a substantial reduction (∼50-fold) in activity against the ʟ-rhamnose-containing polysaccharide (the mutantions did not influence activity against aryl-rhamnosides) [1].

CBM67 members are distributed not only in many bacterial GH78 α-ʟ-rhamnosidases, but also in some Basidiomycete lectins, family 1 pectate lyases, peptidases, and proteins of unknown functions. Protein alignment of candidate members of CBM67 identified five subfamilies within the constructed phylogenetic tree. Although the calcium binding site is conserved in the CBM67, these CBM67 might show different sugar specificity because the ʟ-rhamnose binding residues in SaCBM67 are not retaining in the other CBM67 members. For example, the lectin from Pleurotus cornucopiae contains two CBM67-like sequences in tandem with sequence identities against SaCBM67 of 25 and 35% for the N- and C-terminal modules respectively, shows highest affinity against N-acetyl-ᴅ-galactosamine.

Family Firsts

First Identified
SaCBM67 from the S. avermitilis α-ʟ-rhamnosidase SaRha78A was the first member of the family to be identified and characterized. [1].
First Structural Characterization
The first structure in CBM67 is a module involved in BsRhaB from Bacillus sp. GL1 [2], but the function of the module has not been demonstrated. The first structure-based characterization of a member of family CBM67 was SaCBM67 [1].

References

  1. Fujimoto Z, Jackson A, Michikawa M, Maehara T, Momma M, Henrissat B, Gilbert HJ, and Kaneko S. (2013) The structure of a Streptomyces avermitilis α-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement. J Biol Chem. 288, 12376-85. DOI:10.1074/jbc.M113.460097 | PubMed ID:23486481 | HubMed [Fujimoto2013]
  2. Cui Z, Maruyama Y, Mikami B, Hashimoto W, and Murata K. (2007) Crystal structure of glycoside hydrolase family 78 alpha-L-Rhamnosidase from Bacillus sp. GL1. J Mol Biol. 374, 384-98. DOI:10.1016/j.jmb.2007.09.003 | PubMed ID:17936784 | HubMed [Cui2007]
All Medline abstracts: PubMed | HubMed