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

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.

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.

The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme.