Xyloglucan Is Recognized by Carbohydrate-binding Modules That Interact with β-Glucan Chains

Abstract

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of ␤-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a ␤-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated ␤-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms

“…Expression of all proteins was achieved by adding isopropyl ␤-D-thiogalactopyranoside (1 mM final concentration) to mid-exponential phase (A 600 nm ϭ 0.6) grown cells with incubation for a further 16 h at 19°C. The His 6 -tagged recombinant proteins, and their respective mutant derivatives, were purified from cell-free extracts by immobilized metal affinity chromatography as described previously (28). For crystallization, proteins were further purified by size exclusion chromatography.…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…In addition, several polar residues are capable of making hydrogen bonds with the axial or equatorial O2 of Man or Glc, respectively; while at other sugar-binding subsites, O2 is not a specificity determinant (Charnock et al, 2002a;Bae et al, 2008). CBMs have also been shown to harness both the backbone and side chain of decorated glucans such as xyloglucan (Najmudin et al, 2006). Specificity for this polymer has also been engineered into a xylanspecific module (Gunnarsson et al, 2006), while recent structural information on this protein provides insight into how the observed change in specificity was achieved (Gullfot et al, 2010).…”

The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

“…The outliers in the adsorption profile of the CBMs are the fungal family 1 CBMs (21, 31, 49) as these small (ϳ4 kDa, as compared with 27 kDa for CBM44 and 11 kDa for CBM2a (30,35)) Type A fungal enzyme-derived CBMs are known to preferentially bind to dislocations. These CBMs, which have the highest binding order parameters of all CBMs studied to date (indicating a high preference for crystalline cellulose (48)), appear to localize at dislocations.…”

“…A Type B CBM (CBM44), which contains a cleft-shaped binding site that preferentially binds to amorphous regions of cellulose (30), was used as a probe to determine the amount of accessible amorphous cellulose, whereas a Type A CBM (CBM2a), with a planar, hydrophobic binding face that preferentially binds to crystalline cellulose regions (29), was used as a probe to determine the amount of accessible crystalline cellulose on the dissolving pulp fibers. The amount of each CBM bound to the variably hydrolyzed dissolving pulp over the course of 6 h was used to determine the relative amounts of accessible amorphous and accessible crystalline cellulose (Fig.…”

“…As described earlier by Boraston et al (28), CBMs can be broadly categorized into the three types: Type A, which binds to well ordered (crystalline) substrates via a planar hydrophobic surface composed of tryptophan and tyrosine residues; Type B, which binds via a binding cleft to individual carbohydrate chains; and Type C, which binds via a binding pocket to chain ends and smaller carbohydrate molecules (28). The CBM adsorption technique used in the work described here compares the adsorption of a Type A and a Type B CBM with cellulose where the Type A CBM has been shown to preferentially adsorb to the more crystalline regions of the cellulose surface (29), whereas the Type B CBM preferentially binds to the more amorphous regions (30). Several previous studies have used CBMs with different specificities for crystalline and amorphous regions of the cellulose to try to reveal the differences in surface morphology between the surrounding fiber and the cellulose within fiber dislocations (21,27,(31)(32)(33).…”