A Novel, Noncatalytic Carbohydrate-binding Module Displays Specificity for Galactose-containing Polysaccharides through Calcium-mediated Oligomerization

Abstract

The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to D-galactose and L-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing ␣-and ␤-galactose residues, show that the ligand-binding site in the ␤-sandwich protein is located in the loops that connect the two ␤-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.Carbohydrate protein recognition plays a central role in biology, exemplified by microbe-mediated plant cell wall degradation. The release of sugars from plant cell walls is not only critical for the maintenance of the carbon cycle but is of increasing industrial and environmental significance through the development of second generation lignocellulose-based biofuels (1). The chemical and physical complexity of plant cell walls restricts their accessibility to enzyme attack and thus the recycling of photosynthetically fixed carbon is a relatively slow biological process.

Expression and characteristics of a Ca2+-dependent endoglucanase from Cytophaga hutchinsonii

“…Previous study showed that the cellulose-binding ability of two families of CBMs (CBM36 and CBM60) is Ca 2+ -dependent. It was reported that binding of Ca 2+ changed the conformation of the CBMs to form a circular permutation which can bind to the cellulose substrates (Jamal-Talabani et al 2004;Montanier et al 2011). Unlike other GH9 cellulases, CHU_1280 had only a catalytic domain without recognizable CBM and the binding of Ca 2+ was essential for its cellulose-binding ability and activity.…”

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

“…It has been suggested that the longer ligands adopt a more fixed conformation, through extensive intra-chain hydrogen bonds, which is optimized to recognize the target CBM (39). An alternative possibility is that the CBMs physically associate, resulting in increased affinity for multivalent ligands through avidity effects (15, 40, 41). However, size exclusion chromatography indicated that CBM65A is a monomer (data not shown), although ligand-induced oligomerization is possible; a phenomenon, which would not be observed by studying the molecular mass of the apoprotein (42).…”

“…The general function of CBMs is to direct the cognate catalytic modules to their target substrate within the plant cell wall, thereby increasing the efficiency of catalysis (8–10). The majority of CBMs display a β-sandwich fold with the ligand binding site located in either the concave surface presented by one of the β-sheets, a topography that facilitates the targeting of the internal regions of glycan chains (11–13), or in the loops that connect the two sheets (14, 15). This latter binding site can either target the end (15) or, less frequently, the internal regions of glycan chains (14).…”

“…Binding to crystalline cellulose by Type A CBMs is mediated by a planar hydrophobic surface, which makes apolar contacts with exposed cellulose chains (16). Ligand recognition in CBMs that bind to the internal regions of single polysaccharide chains can be highly specific, exemplified by the CBM6 from the clostridial xylanase Ct Xyn10B, which exclusively targets xylan (14), whereas examples of promiscuous specificity include Cm CBM6 from the Cellvibrio endoglucanase Cm Lic5A that binds to both β1,4-glucans and mixed linked β1,3-β1,4-glucans (17), and the CBM62 from the Cellvibrio xylanase Cj Xyn11A that recognizes β-glucans, xylans, and even β-galactans (15). In both examples plasticity in ligand recognition is achieved through binding to a conserved element of the target glycan, demonstrated by the primary binding site of Cm CBM6, which is specific for cellobiose (Glc-β1,4-Glc), a structure found in both cellulose and the mixed linked glucan (17).…”

Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH‐Π interactions

“…As xyloglucan is an abundant polysaccharide and has an increasing number of industrial applications, finding tools that ease analysis and detection of this branched and heterogeneous polysaccharide are of substantial interest . For the study of xyloglucan, several proteins that can target this polysaccharide have been generated or found in natural sources, for example CBM44 and CBM30, CBM62 from Clostridium thermocellum , or CBM65s derived from Eubacterium cellulosolvens Cel5A . The mode in which xyloglucan is recognized by CBMs can roughly be divided into three categories: (1) CBMs that principally recognize the β‐1,4‐glucose backbone and do not display a definitive preference for xyloglucan over unbranched β‐1,4‐glucose polymers; (2) CBMs that mainly target the branching sugar units of xyloglucan such as CBM62, which interacts with β ‐ d ‐galactoses decorated on the xylose sugars of xyloglucan; (3) CBMs, such as X‐2 L110F and CBM65B of Ec Cel5A, which interact with both the β‐1,4‐glucose backbone and the xylose side chains, reflected by a preference for xyloglucan over unbranched β‐1,4‐glucose polymers.…”