View source for Carbohydrate Binding Module Family 5


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* [[Author]]: ^^^Manjeet Kaur^^^
* [[Responsible Curator]]:  ^^^Appa Rao Podile^^^
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== Ligand specificities ==
The family 5 carbohydrate binding modules (CBM5) have approximately 60 amino acid residues. The CBM5 are found as accessory domains in chitinases <cite>Kezuka2006 Van2000 Uni2012 Manjeet2013</cite>, endoglucanases <cite>Brun1997</cite> and lytic polysaccharide mono-oxygenases (LPMOs) <cite>Forsberg2016 Mutahir2018 Manjeet2019</cite>. This family first originated as a cellulose binding domain family V (CBD V) <cite>Kezuka2006 Simpson1999</cite>. However, CBM5 members have now been reported to have affinity to both cellulose and chitin <cite>Kezuka2006 Van2000 Uni2012 Manjeet2013 Brun1997 Forsberg2016 Mutahir2018 Manjeet2019 Simpson1999</cite>. Deletion of CBM5 from chitinases or LPMOs has shown considerable reduction in chitin binding <cite>Manjeet2013 Forsberg2016 Manjeet2019</cite>. The ''K<sub>d</sub>'' and ''B<sub>max</sub>'' values for CBM5 of an LPMO from ''Cellvibrio japonicas'', ''Cj''LPMO10A for α-chitin, were 5.3 µM and 4.8 µmol/g α-chitin, respectively <cite>Forsberg2016</cite>. The ''K<sub>d</sub>'' values of CBM5 from ''Bacillus thuringiensis'', ''Bt''CBM5 for α- and β-chitin were in the order of 0.6-0.7 µM, whereas the ''B<sub>max</sub>'' value was ~1.9 µmol/g for both α- and β-chitin <cite>Manjeet2019</cite>.

== Structural Features ==
[[File:Figure 1 EcEGZCBM5.png|thumb|300px|right|'''Figure 1.''' CBM5 of an endoglucanase from ''Erwinia chrysanthemi'' [{{PDBlink}}1AIW PDB ID 1AIW] showing surface exposed aromatic residues (Trp18, Trp43 and Tyr44) and a conserved disulfide bond between Cys4 and Cys61.]]
The structures of CBM5 domains have been elucidated for an endoglucanase, CBDEGZ from ''Erwinia chrysanthemi'' (''Ec''EGZCBM5) and two chitinases, ChBDChiB from ''Serratia marcescens'' (''Sm''ChiBCBM5) and ChBDChiC from ''Streptomyces griseus'' HUT6037 (''Sg''ChiCCBM5) <cite>Kezuka2006 Van2000 Brun1997</cite>.

The three structures revealed that CBM5s are composed of five β-strands (β1-5) <cite>Kezuka2006 Van2000 Brun1997</cite>. The β1, β2 and β3 forms the principle structure and the additional short β-strands (β4 and β5) form an antiparallel β-sheet which is independent of the main strand. The ''Ec''EGZCBM5 resembles a ski-boot or L-shaped structure composed of only β-sheets <cite>Brun1997</cite>. Helix structures have not been found in CBM5 modules.

There are a few differences in CBM5 structures from endo-glucanases (''Ec''EGZCBM5) and chitinases (''Sm''ChiBCBM5 and ''Sg''ChiCCBM5). The ''Ec''EGZCBM5 possesses a conserved disulfide bond between Cys4 and Cys61 (Figure 1) <cite>Brun1997</cite>. These disulfide bonds have not been reported in ''Sm''ChiBCBM5 and ''Sg''ChiCCBM5 <cite>Kezuka2006 Van2000</cite>.

CBM5 modules possess surface exposed aromatic residues which interact with polysaccharides most probably through hydrophobic interactions <cite>Kezuka2006 Simpson1999</cite>. These aromatic residues form a flat platform to bind to the planar surfaces of crystalline cellulose/chitin. CBM5 are thus classified under [[Carbohydrate-binding_modules#Types|type A]] CBMs <cite>Gilbert2013</cite>. The ''Ec''EGZCBM5 [{{PDBlink}}1AIW PDB ID 1AIW]  possesses three exposed aromatic residues: Trp18, Trp43 and Tyr44 (Figure 1). Trp18 is present on an extra loop, is linearly aligned to Trp43 and Tyr44 and extends the substrate binding site <cite>Brun1997</cite>. The three residues are essential for complete binding of ''Ec''EGZCBM5. Polar residues like Asp17 are present on the cellulose binding face and form H-bonds to stabilize the appropriate orientation of cellulose binding residues <cite>Brun1997</cite>. Polar residues also form H-bonds with oxygen atoms and/or OH-groups of glucose subunits of cellulose and thus have been proposed to play a role in cellulose-disruption <cite>Brun1997</cite>. Mutation of Asp17 resulted in decreased binding towards cellulose <cite>Simpson1999</cite>.

''Sm''ChiBCBM5 and ''Sg''ChiCCBM5 have only two surface exposed aromatic residues, Trp479 and Trp481 in ''Sm''ChiBCBM5 and Trp59 and Trp60 in ''Sg''ChiCCBM5; whose structural homologues in ''Ec''EGZCBM5 are Trp43 and Tyr44 <cite>Van2000</cite>. The two exposed aromatic residues are sufficient for binding in ''Sm''ChiBCBM5 and ''Sg''ChiCCBM5 <cite>Kezuka2006</cite>. These residues interact extensively and play a vital role in increasing the proximity of substrate through hydrophobic interactions. It has been proposed that either of the two exposed residues should be a tryptophan residue as the Tyr-Tyr pair has not been found in the family <cite>Kezuka2006</cite>.

In ''Sg''ChiCCBM5, six residues (Trp36, Val 48, Tyr 50, Tyr55, Pro66 and Trp72) participate in forming a hydrophobic core in the domain centre. The side chain of Pro66 is internally buried while the remaining 5 residues form the hydrophobic socket <cite>Kezuka2006</cite>. Only two surface exposed aromatic residues (Trp59 and Trp60), which are positioned on a loop between the sheets β2 and β5, are involved in carbohydrate binding  <cite>Kezuka2006</cite>. When protein-substrate interactions were studied between ''Sg''ChiCCBM5 and tri-N-acetyl-chitotriose, it was found that the ligand binding was facilitated by two stacking interactions (Trp59-NAG-1 and Trp-NAG3) and two H-bonds (Trp60-N and NAG2-O7 and Trp56-NE1 and NAG2-O6) <cite>Kezuka2006</cite>.

== Functionalities == 
Multi-modular enzymes like endo-glucanases, chitinases and LPMOs possess CBM5 modules as accessory domains appended to their catalytic domain, either directly or with the help of linkers like FnIII domains <cite>Kezuka2006 Van2000 Uni2012 Manjeet2013 Brun1997 Forsberg2016 Mutahir2018 Manjeet2019</cite>. The CBM5 domains are responsible for increased affinity of these enzymes towards crystalline cellulose or chitin. Their presence also increases the efficiency of enzymes to bind to substrates in a broader pH range <cite>Uni2012 Manjeet2019</cite>. Deletion of the CBM5 resulted in reduction or complete loss of binding in several instances <cite>Manjeet2013</cite>. Deletion of C-terminal FnIII and CBM5 domains from ''Bli''Chi resulted in 5-fold reduction of hydrolytic activity on β-chitin and the mutant was unable to degrade α-chitin <cite>Manjeet2013</cite>. Accessory domains have thus been suggested to play an important role in hydrolysis by moving the enzymes in close proximity of substrates <cite>Manjeet2019</cite>. Presence of CBM5 domains in LPMOs have been shown to alter the product profile while acting on crystalline β-chitin substrates <cite>Manjeet2019</cite>. In ''Bc''LPMO10A, CBM5 promoted substrate binding as well as protected the enzyme from inactivation <cite>Mutahir2018</cite>.

== Family Firsts ==
;First indentified: CBM5 modules were first discovered in Endoglucanase, CBDEGZ from ''Erwinia chrysanthemi'' (''Ec''EGZCBM5) <cite>Brun1997</cite>.
;First structural characterization: The first NMR derived structure of CBM5 was from ''Ec''EGZCBM5 [{{PDBlink}}1AIW PDB ID 1AIW] <cite>Brun1997</cite> and first crystal structure was studied for ChBDChiB from ''Serratia marcescens'' (''Sm''ChiBCBM5) [{{PDBlink}}1E15 PDB ID 1E15] <cite>Van2000</cite>.

== References ==
<biblio>
#Kezuka2006 pmid=16516924
#Van2000 pmid=10823940
#Uni2012 pmid=22451396
#Manjeet2013 pmid=23480960
#Brun1997 pmid=9405041
#Forsberg2016 pmid=26858252
#Mutahir2018 pmid=29993123
#Manjeet2019 pmid=30708015
#Simpson1999 pmid=10419961
#Gilbert2013 pmid=23769966
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM005]]