The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: Structure and biochemistry of the Clostridium thermocellum X6b domain

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" mod ules) that had been implicated in thermostability. We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the C lostridium thermocellum xylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, w ere assayed, and their capacity to bind various carbohydrates and potentiat e hydrolytic activity was determined. The data showed that X6b, but not X6a , increased the activity of the enzyme against insoluble xylan and bound sp ecifically to xylooligosaccharides and various xylans. In contrast, X6a exh ibited no affinity for soluble or insoluble forms of xylan. Isothermal titr ation calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K-d values in th e low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 mu M for xylotriose; and 50 mu M for xylobiose, Negative Delta H and Delta S values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure of X6 b has been solved by X-ray crystallography to a resolution of 2.1 Angstrom. 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-bi nding site. In view of the structural and carbohydrate-binding properties o f X6b, it is proposed that this and related modules be re-assigned as famil y 22 carbohydrate-binding modules.