Single-molecule imaging analysis of binding, processive movement, and dissociation of cellobiohydrolase trichoderma reesei Cel6A and its domains on crystalline cellulose

Akihiko Nakamura, Tomoyuki Tasaki, Daiki Ishiwate, Mayuko Yamamoto, Yasuko Okuni, Akasit Visootsat, Morice Maximilien, Hiroyuki Noji, Taku Uchiyama, Masahiro Samejima, Kiyohiko Igarashi, Ryota Iino (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Ia. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.
Original languageEnglish
Pages (from-to)22404-22413
JournalJournal of Biological Chemistry
Volume291
Issue number43
DOIs
Publication statusPublished - 2016
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Cellulose
Crystalline materials
Imaging techniques
Molecules
Catalytic Domain
Single Molecule Imaging
Cellobiose
Surface diffusion

Keywords

  • cellulase
  • cellulose
  • enzyme kinetics
  • enzyme mechanism
  • microscopic imaging
  • molecular motor
  • processivity
  • protein domain
  • single-molecule biophysics

Cite this

Nakamura, Akihiko ; Tasaki, Tomoyuki ; Ishiwate, Daiki ; Yamamoto, Mayuko ; Okuni, Yasuko ; Visootsat, Akasit ; Maximilien, Morice ; Noji, Hiroyuki ; Uchiyama, Taku ; Samejima, Masahiro ; Igarashi, Kiyohiko ; Iino, Ryota. / Single-molecule imaging analysis of binding, processive movement, and dissociation of cellobiohydrolase trichoderma reesei Cel6A and its domains on crystalline cellulose. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 43. pp. 22404-22413.
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title = "Single-molecule imaging analysis of binding, processive movement, and dissociation of cellobiohydrolase trichoderma reesei Cel6A and its domains on crystalline cellulose",
abstract = "Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Ia. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.",
keywords = "cellulase, cellulose, enzyme kinetics, enzyme mechanism, microscopic imaging, molecular motor, processivity, protein domain, single-molecule biophysics",
author = "Akihiko Nakamura and Tomoyuki Tasaki and Daiki Ishiwate and Mayuko Yamamoto and Yasuko Okuni and Akasit Visootsat and Morice Maximilien and Hiroyuki Noji and Taku Uchiyama and Masahiro Samejima and Kiyohiko Igarashi and Ryota Iino",
year = "2016",
doi = "10.1074/jbc.M116.752048",
language = "English",
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journal = "Journal of Biological Chemistry",
issn = "0021-9258",
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Nakamura, A, Tasaki, T, Ishiwate, D, Yamamoto, M, Okuni, Y, Visootsat, A, Maximilien, M, Noji, H, Uchiyama, T, Samejima, M, Igarashi, K & Iino, R 2016, 'Single-molecule imaging analysis of binding, processive movement, and dissociation of cellobiohydrolase trichoderma reesei Cel6A and its domains on crystalline cellulose', Journal of Biological Chemistry, vol. 291, no. 43, pp. 22404-22413. https://doi.org/10.1074/jbc.M116.752048

Single-molecule imaging analysis of binding, processive movement, and dissociation of cellobiohydrolase trichoderma reesei Cel6A and its domains on crystalline cellulose. / Nakamura, Akihiko; Tasaki, Tomoyuki; Ishiwate, Daiki; Yamamoto, Mayuko; Okuni, Yasuko; Visootsat, Akasit; Maximilien, Morice; Noji, Hiroyuki; Uchiyama, Taku; Samejima, Masahiro; Igarashi, Kiyohiko; Iino, Ryota (Corresponding Author).

In: Journal of Biological Chemistry, Vol. 291, No. 43, 2016, p. 22404-22413.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Single-molecule imaging analysis of binding, processive movement, and dissociation of cellobiohydrolase trichoderma reesei Cel6A and its domains on crystalline cellulose

AU - Nakamura, Akihiko

AU - Tasaki, Tomoyuki

AU - Ishiwate, Daiki

AU - Yamamoto, Mayuko

AU - Okuni, Yasuko

AU - Visootsat, Akasit

AU - Maximilien, Morice

AU - Noji, Hiroyuki

AU - Uchiyama, Taku

AU - Samejima, Masahiro

AU - Igarashi, Kiyohiko

AU - Iino, Ryota

PY - 2016

Y1 - 2016

N2 - Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Ia. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.

AB - Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Ia. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.

KW - cellulase

KW - cellulose

KW - enzyme kinetics

KW - enzyme mechanism

KW - microscopic imaging

KW - molecular motor

KW - processivity

KW - protein domain

KW - single-molecule biophysics

U2 - 10.1074/jbc.M116.752048

DO - 10.1074/jbc.M116.752048

M3 - Article

VL - 291

SP - 22404

EP - 22413

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 43

ER -