Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII

Y Shibafuji, A Nakamura, T Uchihashi, N Sugimoto, S Fukuda, H Watanabe, M Samejima, T Ando, H Noji, Anu Koivula, K Igarashi, R Iino (Corresponding Author)

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Abstract

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Ia and IIII were previously reported as substrates with different crystalline formsanddifferent susceptibilities to hydrolysis byTrCel7A. In this study,weobserved that different susceptibilities of cellulose I a and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Ia and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Ia and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low.Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Ia and IIII and similar fractions of productive binding on cellulose Ia and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Ia and IIII with similar translational rates. With structural models of cellulose Ia and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lane.
Original languageEnglish
Pages (from-to)14056-14065
Number of pages10
JournalJournal of Biological Chemistry
Volume289
Issue number20
DOIs
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Cellulose
Crystalline materials
Imaging techniques
Molecules
Hydrolysis
Enzymes
Single Molecule Imaging
Biofuels
Surface Properties
Atomic Force Microscopy
Fluorescence microscopy
Structural Models
Substrates
Fluorescence Microscopy
Surface properties
Rate constants
Atomic force microscopy

Cite this

Shibafuji, Y ; Nakamura, A ; Uchihashi, T ; Sugimoto, N ; Fukuda, S ; Watanabe, H ; Samejima, M ; Ando, T ; Noji, H ; Koivula, Anu ; Igarashi, K ; Iino, R. / Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 20. pp. 14056-14065.
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title = "Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII",
abstract = "Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Ia and IIII were previously reported as substrates with different crystalline formsanddifferent susceptibilities to hydrolysis byTrCel7A. In this study,weobserved that different susceptibilities of cellulose I a and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Ia and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Ia and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low.Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Ia and IIII and similar fractions of productive binding on cellulose Ia and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Ia and IIII with similar translational rates. With structural models of cellulose Ia and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lane.",
author = "Y Shibafuji and A Nakamura and T Uchihashi and N Sugimoto and S Fukuda and H Watanabe and M Samejima and T Ando and H Noji and Anu Koivula and K Igarashi and R Iino",
year = "2014",
doi = "10.1074/jbc.M113.546085",
language = "English",
volume = "289",
pages = "14056--14065",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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Shibafuji, Y, Nakamura, A, Uchihashi, T, Sugimoto, N, Fukuda, S, Watanabe, H, Samejima, M, Ando, T, Noji, H, Koivula, A, Igarashi, K & Iino, R 2014, 'Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII', Journal of Biological Chemistry, vol. 289, no. 20, pp. 14056-14065. https://doi.org/10.1074/jbc.M113.546085

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII. / Shibafuji, Y; Nakamura, A; Uchihashi, T; Sugimoto, N; Fukuda, S; Watanabe, H; Samejima, M; Ando, T; Noji, H; Koivula, Anu; Igarashi, K; Iino, R (Corresponding Author).

In: Journal of Biological Chemistry, Vol. 289, No. 20, 2014, p. 14056-14065.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII

AU - Shibafuji, Y

AU - Nakamura, A

AU - Uchihashi, T

AU - Sugimoto, N

AU - Fukuda, S

AU - Watanabe, H

AU - Samejima, M

AU - Ando, T

AU - Noji, H

AU - Koivula, Anu

AU - Igarashi, K

AU - Iino, R

PY - 2014

Y1 - 2014

N2 - Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Ia and IIII were previously reported as substrates with different crystalline formsanddifferent susceptibilities to hydrolysis byTrCel7A. In this study,weobserved that different susceptibilities of cellulose I a and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Ia and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Ia and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low.Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Ia and IIII and similar fractions of productive binding on cellulose Ia and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Ia and IIII with similar translational rates. With structural models of cellulose Ia and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lane.

AB - Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Ia and IIII were previously reported as substrates with different crystalline formsanddifferent susceptibilities to hydrolysis byTrCel7A. In this study,weobserved that different susceptibilities of cellulose I a and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Ia and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Ia and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low.Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Ia and IIII and similar fractions of productive binding on cellulose Ia and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Ia and IIII with similar translational rates. With structural models of cellulose Ia and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lane.

U2 - 10.1074/jbc.M113.546085

DO - 10.1074/jbc.M113.546085

M3 - Article

VL - 289

SP - 14056

EP - 14065

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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ER -