Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-␤-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or ⌬4,5-anhydrogalaturonic acid (⌬4,5-GalA). ⌬4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.carbohydrate protein binding ͉ enzyme targeting ͉ plant cell wall degradation ͉ protein cell attachment ͉ X-ray crystallography

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.

The enzymatic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The enzymes that catalyze this process include xylanases that degrade xylan, a ␤-1,4-xylose polymer that is decorated with various sugars. Although xylanases efficiently hydrolyze unsubstituted xylans, these enzymes are unable to access highly decorated forms of the polysaccharide, such as arabinoxylans that contain arabinofuranose decorations. Here, we show that a Clostridium thermocellum enzyme, designated CtXyl5A, hydrolyzes arabinoxylans but does not attack unsubstituted xylans. Analysis of the reaction products generated by CtXyl5A showed that all the oligosaccharides contain an O3 arabinose linked to the reducing end xylose. The crystal structure of the catalytic module (CtGH5) of CtXyl5A, appended to a family 6 noncatalytic carbohydrate-binding module (CtCBM6), showed that CtGH5 displays a canonical (␣/␤) 8 -barrel fold with the substrate binding cleft running along the surface of the protein. The catalytic apparatus is housed in the center of the cleft. Adjacent to the ؊1 subsite is a pocket that could accommodate an L-arabinofuranose-linked ␣-1,3 to the active site xylose, which is likely to function as a key specificity determinant. CtCBM6, which adopts a ␤-sandwich fold, recognizes the termini of xylo-and gluco-configured oligosaccharides, consistent with the pocket topology displayed by the ligand-binding site. In contrast to typical modular glycoside hydrolases, there is an extensive hydrophobic interface between CtGH5 and CtCBM6, and thus the two modules cannot function as independent entities.

Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall–degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated Ct Cel124. The protein was shown to be an endo -acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo -cellulase. The crystal structure of Ct Cel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose.

The deconstruction of the plant cell wall is an important biological process that is attracting considerable industrial interest, particularly in the bioenergy sector. Enzymes that attack the plant cell wall generally contain one or more noncatalytic carbohydrate binding modules (CBMs) that play an important targeting function. While CBMs that bind to the backbones of plant structural polysaccharides have been widely described, modules that recognize components of the vast array of decorations displayed on these polymers have been relatively unexplored. Here we show that a family 35 CBM member (CBM35), designated CtCBM35-Gal, binds to alpha-D-galactose (Gal) and, within the context of the plant cell wall, targets the alpha-1,6-Gal residues of galactomannan but not the beta-D-Gal residues in xyloglucan. The crystal structure of CtCBM35-Gal reveals a canonical beta-sandwich fold. Site-directed mutagenesis studies showed that the ligand is accommodated within the loops that connect the two beta-sheets. Although the ligand binding site of the CBM displays significant structural similarity with calcium-dependent CBM35s that target uronic acids, subtle differences in the conformation of conserved residues in the ligand binding site lead to the loss of metal binding and uronate recognition. A model is proposed in which the orientation of the pair of aromatic residues that interact with the two faces of the Gal pyranose ring plays a pivotal role in orientating the axial O4 atom of the ligand toward Asn140, which is invariant in CBM35. The ligand recognition site of exo-CBM35s (CBM35-Gal and the uronic acid binding CBM35s) appears to overlap with that of CBM35-Man, which binds to the internal regions of mannan, a beta-polymer of mannose. Using site-directed mutagenesis, we show that although there is conservation of several functional residues within the binding sites of endo- and exo-CBM35s, the endo-CBM does not utilize Asn113 (equivalent to Asn140 in CBM35-Gal) in mannan binding, despite the importance of the equivalent residue in ligand recognition across the CBM35 and CBM6 landscape. The data presented in this report are placed within a wider phylogenetic context for the CBM35 family.