title: 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.
abstract: 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 A and 2.0 A 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 alpha-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 hydrolases.
authors.affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, Y010 5DD, UK.
authors.forename: E
authors.initials: E
authors.lastname: Sabini
authors.forename: G
authors.initials: G
authors.lastname: Sulzenbacher
authors.forename: M
authors.initials: M
authors.lastname: Dauter
authors.forename: Z
authors.initials: Z
authors.lastname: Dauter
authors.forename: P L
authors.initials: PL
authors.lastname: Jørgensen
authors.forename: M
authors.initials: M
authors.lastname: Schülein
authors.forename: C
authors.initials: C
authors.lastname: Dupont
authors.forename: G J
authors.initials: GJ
authors.lastname: Davies
authors.forename: K S
authors.initials: KS
authors.lastname: Wilson
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D020134:Catalytic Domain; D006027:Glycosides / Q000378:metabolism*; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000378:metabolism*
chemical_list: D006027:Glycosides; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase