Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps
Abstract
Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor’s mechanistic action has yet to be revealed. We develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Moreover, through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A’s molecular motility mechanism.
- Authors:
-
- Vanderbilt Univ., Nashville, TN (United States)
- National Univ. of Singapore (Singapore)
- State Univ. of New Jersey, Piscataway, NJ (United States)
- Vanderbilt Univ., Nashville, TN (United States); National Univ. of Singapore (Singapore)
- Publication Date:
- Research Org.:
- Michigan State Univ., East Lansing, MI (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1240602
- Grant/Contract Number:
- FC02-07ER64494
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 6; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Brady, Sonia K., Sreelatha, Sarangapani, Feng, Yinnian, Chundawat, Shishir P. S., and Lang, Matthew J. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps. United States: N. p., 2015.
Web. doi:10.1038/ncomms10149.
Brady, Sonia K., Sreelatha, Sarangapani, Feng, Yinnian, Chundawat, Shishir P. S., & Lang, Matthew J. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps. United States. https://doi.org/10.1038/ncomms10149
Brady, Sonia K., Sreelatha, Sarangapani, Feng, Yinnian, Chundawat, Shishir P. S., and Lang, Matthew J. Thu .
"Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps". United States. https://doi.org/10.1038/ncomms10149. https://www.osti.gov/servlets/purl/1240602.
@article{osti_1240602,
title = {Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps},
author = {Brady, Sonia K. and Sreelatha, Sarangapani and Feng, Yinnian and Chundawat, Shishir P. S. and Lang, Matthew J.},
abstractNote = {Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor’s mechanistic action has yet to be revealed. We develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Moreover, through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A’s molecular motility mechanism.},
doi = {10.1038/ncomms10149},
journal = {Nature Communications},
number = ,
volume = 6,
place = {United States},
year = {Thu Dec 10 00:00:00 EST 2015},
month = {Thu Dec 10 00:00:00 EST 2015}
}
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Figures / Tables:
Figure 1: Constructs and assay schematic. Construct details and optical trap assay schematic for (a) wtTrCel7A, where a DNA-bound sulfo-SMCC crosslinks through available surface lysines (scale bar, 1 nm), (b) isolated biotin-labelled CD ligated to DNA through a ½ anti-biotin antibody and (c) isolated CBM tethered through a DNA-bound anti-Hismore »
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