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Carbohydrate Binding Module Family 44
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CBM44, from the CtCel9D-Cel44A enzyme produced by Acetivibrio thermocellus (formerly Clostridium thermocellum), targets ß-1,4-polymers such as xyloglucan and cellulose (hydroxyethylcellulose and Avicel), mixed linkage ß-1,3/ß-1,4- glucans (lichenan and barley) or glucomannan (konjac)  and is classified as a type B CBM. Affinity for xylan was very low and binding to laminarin, curdlan, pullulan, pustulan, galactomannan or galactan was negative. Isothermal titration calorimetry (ITC) revealed highest affinity for xyloglucan as a polysaccharide (~81.6 x 104 M-1), which was comparable to the affinity for cellohexaose as an oligosaccharide (~72.8 x 104 M-1). For other cello-oligosaccharides, this affinity decreased with decreasing chain length, while no binding was detected for cellotriose .
Like many CBMs, CBM44 exhibits a typical ß-sandwich fold: two antiparallel ß-sheets form a concave and a convex surface. The concave surface forms a deep hydrophobic ligand-binding cleft that is estimated to accommodate up to five glucose residues (~24 Å). Here, three tryptophans (W189, W194 and W198) act as key residues to mediate ligand binding (Figure 1), as confirmed by affinity gel electrophoresis (AGE) and ITC analyses of specific mutants . The orientation of the tryptophans corresponds to the slightly twisted conformation of cello-oligosaccharides in solution .
Carbohydrate binding and targeting were described as functions of CBM44. While carbohydrate binding was confirmed by AGE and ITC, the targeting effect was analyzed by the ability of CBM44 to maintain enzyme activity of GH44 (Cel44A) exposed to a substrate mixture . The ensemble of a GH44 and a CBM44 was found in the multimodular cellulase CtCel9D-Cel44A from Acetivibrio thermocellus and ligand spectra of both modules are consistent . In the targeting experiment, the fused GH44-CBM44 construct was compared to the truncated GH44. In separate xyloglucan or carboxymethylcellulose incubations, the activity of GH44-CBM44 was comparable to that of GH44 alone. In a next step, substrate mixtures were used. These mixtures also contained laminarin and pustulan, which were not degraded. While GH44 activity was decreased, activity was restored by GH44-CBM44. Thus, given the complex carbohydrates present in plant cell walls, CBM44 may guide the enzyme to its target sugars. Such effects have also been achieved in synthetic CBM44-enzyme chimeras, e.g. GH12 or GH28 [3, 4].
- First Identified
- CBM44 was first described as an unknown C-terminal domain in the multimodular cellulase CtCel9D-Cel44A in Acetivibrio thermocellus, formerly designated as CelJ and as Clostridium thermocellum [5, 6]. The protein also features two catalytic domains (GH44 and GH9), a CBM30 and an internal dockerin to target the protein to the cellulosome. The C-terminal region was then identified as having a PKD module as well as a novel CBM binding ß-1,4-glucans, hereby founding the family 44 .
- First Structural Characterization
- The same CBM44, together with the preceding PKD module, represents the first structurally characterized candidate of this family (PDB 2c26) .
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Sugiyama H, Hisamichi K, Usui T, Sakai K, Ishiyama J (2000). A study of the conformation of beta-1,4-linked glucose oligomers, cellobiose to cellohexaose, in solution. J Mol Struct. 2000;556(1-3):173-7. DOI: 10.1016/S0022-2860(00)00630-X.
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Carli S, Meleiro LP, Salgado JCS, Ward RJ (2022). Synthetic carbohydrate-binding module-endogalacturonase chimeras increase catalytic efficiency and saccharification of lignocellulose residues. Biomass Conv Bioref. 2022. DOI: 10.1007/s13399-022-02716-6.
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