The Dissociation Mechanism of Processive Cellulases
Abstract
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.
- Authors:
-
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Univ. of Tartu, Tartu (Estonia)
- Swedish Univ. of Agricultural Sciences, Uppsala (Sweden)
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
- OSTI Identifier:
- 1574196
- Report Number(s):
- NREL/JA-2700-74624
Journal ID: ISSN 0027-8424
- Grant/Contract Number:
- AC36-08GO28308
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Proceedings of the National Academy of Sciences of the United States of America
- Additional Journal Information:
- Journal Volume: 116; Journal Issue: 46; Journal ID: ISSN 0027-8424
- Publisher:
- National Academy of Sciences
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; biofuels; cellulases; molecular mechanism; molecular simulation; crystal structure; BCPL
Citation Formats
Vermaas, Joshua, Kont, Riin, Beckham, Gregg T, Crowley, Michael F, Gudmundsson, Mikael, Sandgren, Mats, Stahlberg, Jerry, Valjamae, Priit, and Knott, Brandon C. The Dissociation Mechanism of Processive Cellulases. United States: N. p., 2019.
Web. doi:10.1073/pnas.1913398116.
Vermaas, Joshua, Kont, Riin, Beckham, Gregg T, Crowley, Michael F, Gudmundsson, Mikael, Sandgren, Mats, Stahlberg, Jerry, Valjamae, Priit, & Knott, Brandon C. The Dissociation Mechanism of Processive Cellulases. United States. https://doi.org/10.1073/pnas.1913398116
Vermaas, Joshua, Kont, Riin, Beckham, Gregg T, Crowley, Michael F, Gudmundsson, Mikael, Sandgren, Mats, Stahlberg, Jerry, Valjamae, Priit, and Knott, Brandon C. Tue .
"The Dissociation Mechanism of Processive Cellulases". United States. https://doi.org/10.1073/pnas.1913398116. https://www.osti.gov/servlets/purl/1574196.
@article{osti_1574196,
title = {The Dissociation Mechanism of Processive Cellulases},
author = {Vermaas, Joshua and Kont, Riin and Beckham, Gregg T and Crowley, Michael F and Gudmundsson, Mikael and Sandgren, Mats and Stahlberg, Jerry and Valjamae, Priit and Knott, Brandon C},
abstractNote = {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.},
doi = {10.1073/pnas.1913398116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 46,
volume = 116,
place = {United States},
year = {Tue Nov 12 00:00:00 EST 2019},
month = {Tue Nov 12 00:00:00 EST 2019}
}
Web of Science
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