The X6 “Thermostabilizing” Domains of Xylanases Are Carbohydrate-Binding Modules:  Structure and Biochemistry of the Clostridium thermocellum X6b Domain^,

Abstract

Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknown function (termed "X6" modules) that had been implicated in thermostability. We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined. The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 microM for xylotriose; and 50 microM for xylobiose. Negative DeltaH and DeltaS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 A. The protein is a beta-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re-assigned as family 22 carbohydrate-binding modules.

X4 Modules Represent a New Family of Carbohydrate-binding Modules That Display Novel Properties

“…8 and Table III). The ⌬H and T⌬S values for the binding of the CBM35 to oat-spelt xylan are negative, similar to Man5C-CBM35 and other CBMs that interact with soluble polysaccharides (10,11,15). Titration of the apo form of Abf62A-CBM35 with calcium demonstrates that the protein binds tightly to the divalent metal ion (Fig.…”

“…Previous studies have shown that CBMs from families 4, 6, 9, and 22 contain one or more calcium ions located at sites remote from the ligand binding cleft, suggesting a structural role for the metal (10,29,(33)(34)(35). In support of this view, removal of calcium from both cellulose and xylan-binding CBM4s reduced the temperature at which the proteins unfolded by 8°C (34) and 23°C (35), respectively.…”

“…The polysaccharide chains in DGM are likely to associate in a less ordered fashion than those found in crystalline mannans and are therefore analogous to amorphous cellulose where discrete polysaccharide chains are able to interact with CBMs that contain a ligand binding cleft (18). Thus, Man5C-CBM35 appears to exhibit features typical of Type B CBMs, which accommodate individual polysaccharide chains in a binding site that displays a cleft topology (10,15), but are unable to interact with the flat surfaces of highly crystalline ligands such as ivory nut mannan.…”

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…increasing the concentration of the enzyme on the surface of the substrate) leads to more rapid degradation of the polysaccharide [22]. There are numerous examples in the literature where proteolytic excision or genetic truncation of CBMs from the catalytic modules results in significant decreases in the activity of the enzymes on insoluble, but not soluble polysaccharides (see [2,[22][23][24][25][26][27][28][29] for examples). It should be pointed out that there are examples of CBMs that have become components of the substrate-binding sites of glycoside hydrolases, and are pivotal to the substrate specificity and mode of action of the cognate enzymes.…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Ligand recognition by these CBMs is driven primarily through an increase in entropy, resulting in the desolvation of the interacting macromolecules (Creagh et al, 1996). By contrast, enthalpy drives the binding of CBMs to discreet polysaccharide chains, where both polar and apolar interactions occur, while entropy has a negative impact on overall affinity (Charnock et al, 2000(Charnock et al, , 2002aBolam et al, 2001;Boraston et al, 2002aBoraston et al, , 2002b. The negative entropy may reflect conformational restriction of the ligand bound to the protein, which is not entirely offset by the release of tightly bound water molecules (for review, see Boraston et al, 2004).…”