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)

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19 Citations (Scopus)

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

Keywords

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

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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, 291(43), 22404-22413. https://doi.org/10.1074/jbc.M116.752048