Binding Specificity and Thermodynamics of a Family 9 Carbohydrate-Binding Module from Thermotoga maritima Xylanase 10A

Abstract

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.

Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

“…CBM9 binds to the reducing end of cellulose and xylan chains and thus provides a direct readout of cellulose hydrolysis (13). The Calcofluor White staining data (Fig.…”

“…During a titration experiment, the protein sample at 88 μM was injected with 20 × 2 μL aliquots of 2 mM cellohexaose. For experiments with RC, prepared as described previously (13), the ligand was in the cell at 29.4 mg∕mL, and the protein (835 μM) was the titrant. Integrated heat effects, after correction for heats of dilution, were analyzed by nonlinear regression using a single site-binding model (Microcal Origin, version 2.9).…”

Substrate binding by a bacterial ABC transporter involved in polysaccharide export

“…The aligned sequences in SI Fig. 7 were scored for conservation by using ConSurf (43,44) ars of xylans and cellulose (14,15). Similarily, the CBMs of laminarinase from Bacillus halodurans, and ␤-agarase from Saccharophagus degradans, specifically recognize the nonreducing terminus of their carbohydrate substrates (16,17).…”

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…It is now becoming apparent that this targeting function is even more subtle than the somewhat crude partitioning of enzymes to the different polysaccharides of plant cell walls. The family 9 CBM from xylanase 10A of Thermotoga maritima has a distinct specificity for only the reducing ends of polysaccharides, suggesting the intriguing notion of targeting to damaged regions of plant cell walls [32,33]. An elegant study by Carrard et al [34] showed that family 1 and family 3 CBMs that were appended to the same catalytic module displayed different capacities to degrade crystalline cellulose, implying that these non-catalytic modules can recognize distinct regions of this otherwise chemically invariant polysaccharide.…”

“…WGA) were first discovered as lectins with small-sugar-binding activity and have only subsequently been included as CBMs due to their discovery in a number of glycoside hydrolases. The only characterized member of family 9 is the C-terminal CBM from T. maritima xylanase 10A [32,48,64]. In general, this family of CBMs is found exclusively in xylanases, and this particular CBM from T. maritima has the remarkable property of recognizing the reducing end sugars of xylans and cellulose.…”