Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei

Structure. 1999 Sep 15;7(9):1035-45. doi: 10.1016/s0969-2126(99)80171-3.

Abstract

Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops.

Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG).

Conclusions: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Catalysis
  • Cellulase / chemistry*
  • Cellulase / genetics
  • Cellulase / metabolism*
  • Cellulose 1,4-beta-Cellobiosidase
  • Crystallography, X-Ray
  • Glucosides / chemistry
  • Glucosides / metabolism
  • Ligands
  • Mutation
  • Protein Conformation
  • Structure-Activity Relationship
  • Trichoderma / enzymology*

Substances

  • Glucosides
  • Ligands
  • iodobenzyl-glucopyranosyl-(1,4)-xylopyranoside
  • Cellulase
  • Cellulose 1,4-beta-Cellobiosidase

Associated data

  • PDB/1QJW
  • PDB/1QK0
  • PDB/1QK2