Crystallographic analysis shows substrate binding at the -3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-beta-xylanases

Biochem J. 2008 Feb 15;410(1):71-9. doi: 10.1042/BJ20071128.

Abstract

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein-ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus niger / enzymology
  • Bacillus subtilis / enzymology
  • Base Sequence
  • Binding Sites
  • Crystallography, X-Ray
  • DNA Primers
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / metabolism*
  • Polymerase Chain Reaction
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • DNA Primers
  • Recombinant Proteins
  • Glycoside Hydrolases