Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 46, Pages 23061-23067Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1913398116
Keywords
biofuels; cellulases; molecular mechanism; molecular simulation; crystal structure
Categories
Funding
- US Department of Energy (DOE) [DE-AC36-08GO28308]
- US DOE, Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office
- Estonian Science Foundation [PUT1024]
- NSF at the Texas Advanced Computing Center (Stampede2) [ACI-1548562, MCB-090159]
- DOE Office of EERE [DE-AC36-08GO28308]
- Early Science program on Eagle
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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.
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