Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study.

The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods. CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose. The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids. Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft. In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs. The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces.

Crystal structures of the family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A in native and ligand-bound forms.

The C-terminal module of the thermostable Thermotoga maritima xylanase 10A (CBM9-2) is a family 9 carbohydrate-binding module that binds to amorphous and crystalline cellulose and a range of soluble di- and monosaccharides as well as to cello and xylo oligomers of different degrees of polymerization [Boraston, A. B., Creagh, A. L., Alam, Md. M., Kormos, J. M., Tomme, P., Haynes, C. A., Warren, R. A. J., and Kilburn, D. G. (2001) Biochemistry 40, 6240-6247]. The crystal structure of CBM9-2 has been determined by the multiwavelength anomalous dispersion method to 1.9 A resolution. CBM9-2 assumes a beta-sandwich fold and contains three metal binding sites. The bound metal atoms, which are most likely calcium cations, are in an octahedral coordination. The crystal structures of CBM9-2 in complex with glucose and cellobiose were also determined in order to identify the sugar-binding site and provide insight into the structural basis for sugar binding by CBM9-2. The sugar-binding site is a solvent-exposed slot sufficient in depth, width, and length to accommodate a disaccharide. Two tryptophan residues are stacked together on the surface of the protein forming the sugar-binding site. From the complex structures with glucose and cellobiose, it was inferred that CBM9-2 binds exclusively to the reducing end of mono-, di-, and oligosaccharides with an intricate hydrogen-bonding network involving mainly charged residues, as well as stacking interactions by Trp175 and Trp71. The binding interactions are limited to disaccharides as was expected from calorimetric data. Comparison of the glucose and cellobiose complexes revealed surprising differences in binding of these two substrates by CBM9-2. Cellobiose was found to bind in a distinct orientation from glucose, while still maintaining optimal stacking and electrostatic interactions with the reducing end sugar.

Detailed structural analysis of glycosidase/inhibitor interactions: complexes of Cex from Cellulomonas fimi with xylobiose-derived aza-sugars.

Detailed insights into the mode of binding of a series of tight-binding aza-sugar glycosidase inhibitors of two fundamentally different classes are described through X-ray crystallographic studies of complexes with the retaining family 10 xylanase Cex from Cellulomonas fimi. Complexes with xylobiose-derived aza-sugar inhibitors of the substituted "amidine" class (xylobio-imidazole, K(i) = 150 nM; xylobio-lactam oxime, K(i) = 370 nM) reveal lateral interaction of the "glycosidic" nitrogen with the acid/base catalyst (Glu127) and hydrogen bonding of the sugar 2-hydroxyl with the catalytic nucleophile (Glu233), as expected. Tight binding of xylobio-isofagomine (K(i) = 130 nM) appears to be a consequence of strong interactions of the ring nitrogen with the catalytic nucleophile while, surprisingly, no direct protein contacts are made with the ring nitrogen of the xylobio-deoxynojirimycin analogue (K(i) = 5800 nM). Instead the nitrogen interacts with two ordered water molecules, thereby accounting for its relatively weaker binding, though it still binds some 1200-fold more tightly than does xylobiose, presumably as a consequence of electrostatic interactions at the active site. Dramatically weaker binding of these same inhibitors to the family 11 xylanase Bcx from Bacillus circulans (K(i) from 0.5 to 1.5 mM) is rationalized for the substituted amidines on the basis that this enzyme utilizes a syn protonation trajectory and likely hydrolyzes via a (2,5)B boat transition state. Weaker binding of the deoxynojirimycin and isofagomine analogues likely reflects the energetic penalty for distortion of these analogues to a (2,5)B conformation, possibly coupled with destabilizing interactions with Tyr69, a conserved, catalytically essential active site residue.

Insights into transition state stabilization of the beta-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants.

The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of considerable interest and debate for many years. The visualization of a covalent glycosyl enzyme intermediate by X-ray crystallography was first accomplished with a saccharide substrate substituted with fluorine at its 2-position. The structure implicated major roles for residue His 205 and for the 2-hydroxyl position of the proximal saccharide in binding and catalysis. Here we have studied the kinetic behavior of various His 205 mutants. One of these mutants, a double mutant H205N/E127A, has been used to stabilize a covalent glycosyl-enzyme intermediate involving an unsubstituted sugar, permitting crystallographic analysis of the interactions between its 2-hydroxyl group and the enzyme.

Exploring the cellulose/xylan specificity of the beta-1,4-glycanase cex from Cellulomonas fimi through crystallography and mutation.

The retaining beta-1,4-glycanase Cex from Cellulomonas fimi, a family 10 glycosyl hydrolase, hydrolyzes xylan 40-fold more efficiently than cellulose. To gain insight into the nature of its preference for xylan, we determined the crystal structure of the Cex catalytic domain (Cex-cd) trapped as its covalent 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate to 1.9 A resolution. Together with the crystal structure of unliganded Cex-cd [White, A., et al. (1994) Biochemistry 33, 12546-12552] and the previously determined crystal structure of the covalent 2-deoxy-2-fluorocellobiosyl-Cex-cd intermediate [White, A., et al. (1996) Nat. Struct. Biol. 3, 149-154], this structure provides a convincing rationale for the observed substrate specificity in Cex. Two active site residues, Gln87 and Trp281, are found to sterically hinder the binding of glucosides and must rearrange to accommodate these substrates. Such rearrangements are not necessary for the binding of xylobiosides. The importance of this observation was tested by examining the catalytic behavior of the enzyme with Gln87 mutated to Met. This mutation had no measurable effect on substrate affinity or turnover number relative to the wild type enzyme, indicating that the Met side chain could accommodate the glucoside moiety as effectively as the wild type Gln residue. Subsequent mutagenesis studies will address the role of entropic versus enthalpic contributions to binding by introducing side chains that might be more rigid in the unliganded enzyme.