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

Background: The enzymatic hydrolysis of glycosides involves the formation a nd subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxoc arbenium-ion-like transition states. The covalent intermediate may be trapp ed on-enzyme using 2-fluoro-substituted glycosides, which provide details o f 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 t he amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, ve rsus glucose-based, substrates. Results: The structure of the family 11 xylanase, Xyl11, from Bacillus agar adhaerens has been solved using X-ray crystallography in both native and xy lobiosyl-enzyme intermediate forms at 1.78 Angstrom and 2.0 Angstrom resolu tion, respectively The covalent glycosyl-enzyme intermediate has been trapp ed using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike cov alent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual B -2,B-5 conformation. Conclusions: The 2,5B conformation found for the a-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the C-4(1) chair conformation observed for o ther systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covale nt glycosyl-enzyme intermediate with the equivalent structure for the relat ed family 12 endoglucanase, CelB, from Streptomyces lividans reveals the li kely determinants for substrate specificity in this dan of glycoside hydrol ases.