The Dissociation Mechanism of Processive Cellulases

Brandon Knott, Gregg Beckham, Michael Crowley, Riin Kont, Mikael Gudmundsson, Mats Sandgren, Jerry Stahlberg, Priit Valjamae, Joshua Vermaas

Research output: Contribution to journalArticlepeer-review

33 Scopus Citations


Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Original languageAmerican English
Pages (from-to)23061-23067
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number46
StatePublished - 12 Nov 2019

Bibliographical note

Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.

NREL Publication Number

  • NREL/JA-2700-74624


  • Biofuels
  • Cellulases
  • Crystal structure
  • Molecular mechanism
  • Molecular simulation


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