Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase Original Research Article

Background The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications — in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. Results The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 Å and 2.0 Å resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. Conclusions The 2,5B conformation found for the α-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside