Visualization of Functional Structure and Kinetic Dynamics of Cellulases

  • Akihiko NakamuraEmail author
  • Ryota Iino
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1104)


Cellulose is the most abundant carbohydrate on earth and hydrolyzed by cellulases in nature. During catalysis, cellulase transfers protons to and from the oxygen atoms of the glycosidic bond and a water molecule. Since cellulose is an insoluble polymer, some kinds of cellulases, with high activity toward crystalline cellulose, move on the crystal surface with continuous hydrolysis of the molecular chain. In addition, binding and dissociation on/from the crystal surface are also important elementary steps of the reaction cycle. Recently, these interesting features of cellulases can be directly analyzed, due to the development of visualization techniques. In this chapter, we introduce (1) visualization of the protonation state of the catalytic residue by neutron crystallography, (2) visualization of processive movement on the crystal surface by high-speed atomic force microscopy, and (3) visualization of binding and dissociation events by single-molecule fluorescence microscopy.


Cellulase cellulose Processivity Molecular motor Single-molecule analysis Neutron crystallography Proton pathway 



This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan [grant numbers JP17K19213, JP16H00789, JP16H00858, and JP15H04366 to R. I., JP15H06898, JP17K18429, and JP17H05899 to A. N.], Advanced Technology Institute Research Grants 2015 (RG2709 to A.N.).


  1. Ando T (2013) High-speed atomic force microscopy (AFM). In: Encyclopedia of biophysics. vol Chapter 478. Springer, Berlin/Heidelberg/Berlin/Heidelberg, pp 984–987. CrossRefGoogle Scholar
  2. Ando T (2017) Directly watching biomolecules in action by high-speed atomic force microscopy. Biophys Rev 9(4):421–429. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Brady SK, Sreelatha S, Feng Y, Chundawat SPS, Lang MJ (2015) Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps. Nat Commun 6:10149. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chanzy H, Henrissat B (1985) Undirectional degradation of valonia cellulose microcrystals subjected to cellulase action. FEBS Lett 184(2):285–288. CrossRefGoogle Scholar
  5. Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3(9):853–859. CrossRefPubMedGoogle Scholar
  6. Igarashi K, Koivula A, Wada M, Kimura S, Penttila M, Samejima M (2009) High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose. J Biol Chem 284(52):36186–36190. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Igarashi K, Uchihashi T, Koivula A, Wada M, Kimura S, Okamoto T, Penttila M, Ando T, Samejima M (2011) Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface. Science 333(6047):1279–1282. CrossRefPubMedGoogle Scholar
  8. Igarashi K, Uchihashi T, Uchiyama T, Sugimoto H, Wada M, Suzuki K, Sakuda S, Ando T, Watanabe T, Samejima M (2014) Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin. Nat Commun 5:1–7. CrossRefGoogle Scholar
  9. Iino R, Iida T, Nakamura A, E-i S, You H, Sako Y (2017) Single-molecule imaging and manipulation of biomolecular machines and systems. Biochim Biophys Acta S0304-4165(17):30253–30252. CrossRefGoogle Scholar
  10. Imai T, Boisset C, Samejima M, Igarashi K, Sugiyama J (1998) Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals. FEBS Lett 432(3):113–116. CrossRefPubMedGoogle Scholar
  11. Isojima H, Iino R, Niitani Y, Noji H, Tomishige M (2016) Direct observation of intermediate states during the stepping motion of kinesin-1. Nat Chem Biol 12(4):290–297. CrossRefPubMedGoogle Scholar
  12. Jalak J, Kurasin M, Teugjas H, Valjamae P (2012) Endo-exo synergism in cellulose hydrolysis revisited. J Biol Chem 287(34):28802–28815. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Julliand V, de Vaux A, Millet L, Fonty G (1999) Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum. Appl Environ Microbiol 65(8):3738–3741PubMedPubMedCentralGoogle Scholar
  14. Kitamura K, Tokunaga M, Iwane AH, Yanagida T (1999) A single myosin head moves along an actin filament with regular steps of 5.3 nanometres. Nature 397(6715):129–134. CrossRefPubMedGoogle Scholar
  15. Kodera N, Uchida K, Ando T, Aizawa S-I (2015) Two-ball structure of the flagellar hook-length control protein FliK as revealed by high-speed atomic force microscopy. J Mol Biol 427(2):406–414. CrossRefPubMedGoogle Scholar
  16. Kodera N, Yamamoto D, Ishikawa R, Ando T (2010) Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468(7320):72–76. CrossRefPubMedGoogle Scholar
  17. Kusaka K, Hosoya T, Yamada T, Tomoyori K, Ohhara T, Katagiri M, Kurihara K, Tanaka I, Niimura N (2013) Evaluation of performance for IBARAKI biological crystal diffractometer iBIX with new detectors. J Synchrotron Radiat 20(6):994–998. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Leschine SB (1995) Cellulose degradation in anaerobic environments. Annu Rev Microbiol 49(1):399–426. CrossRefPubMedGoogle Scholar
  19. Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42(D1):D490–D495. CrossRefPubMedGoogle Scholar
  20. McCarter JD, Stephen Withers G (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol 4(6):885–892. CrossRefPubMedGoogle Scholar
  21. McMaster WH, Del Grande NK, Mallett JH, Hubbell JH (1969) Compilation of X-ray cross sections. Lawrence Livermore National Laboratory Report UCRL-50174Google Scholar
  22. Nakamura A, Ishida T, Fushinobu S, Kusaka K, Tanaka I, Inaka K, Higuchi Y, Masaki M, Ohta K, Kaneko S, Niimura N, Igarashi K, Samejima M (2013a) Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis. J Synchrotron Radiat 20(6):859–863. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Nakamura A, Ishida T, Kusaka K, Yamada T, Fushinobu S, Tanaka I, Kaneko S, Ohta K, Tanaka H, Inaka K, Higuchi Y, Niimura N, Samejima M, Igarashi K (2015) “Newton’s cradle” proton relay with amide-imidic acid tautomerization in inverting cellulase visualized by neutron crystallography. Sci Adv 1(7):e1500263–e1500263. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Nakamura A, Tasaki T, Ishiwata 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. J Biol Chem 291(43):22404–22413. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Nakamura A, Tasaki T, Okuni Y, Song C, Murata K, Kozai T, Hara M, Sugimoto H, Suzuki K, Watanabe T, Uchihashi T, Noji H, Iino R (2018) Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis. Phys Chem Chem Phys 20(5):3010–3018. CrossRefPubMedGoogle Scholar
  26. Nakamura A, Tsukada T, Auer S, Furuta T, Wada M, Koivula A, Igarashi K, Samejima M (2013b) The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose. J Biol Chem 288(19):13503–13510. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Nakamura A, Watanabe H, Ishida T, Uchihashi T, Wada M, Ando T, Igarashi K, Samejima M (2014) Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose. J Am Chem Soc 136(12):4584–4592. CrossRefPubMedGoogle Scholar
  28. Ogata H, Nishikawa K, Lubitz W (2015) Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase. Nature 520(7548):571–574. CrossRefPubMedGoogle Scholar
  29. Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE II, Sandgren M, Ståhlberg J, Stals I, Tan Z, Beckham GT (2013) Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci U S A 110(36):14646–14651. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Shibafuji Y, Nakamura A, Uchihashi T, Sugimoto N, Fukuda S, Watanabe H, Masahiro S, Toshio A, Hiroyuki N, Koivula A, Kiyohiko I, Ryota I (2014) Single-molecule imaging analysis of elementary reaction steps of Trichoderma Reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII. J Biol Chem 289(20):14056–14065. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Shibata M, Yamashita H, Uchihashi T, Kandori H, Ando T (2010) High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin. Nat Nanotechnol 5(3):208–212. CrossRefPubMedGoogle Scholar
  32. Štursová M, Žifčáková L, Leigh MB, Burgess R, Baldrian P (2012) Cellulose utilization in forest litter and soil: identification of bacterial and fungal decomposers. FEMS Microbiol Ecol 80(3):735–746. CrossRefPubMedGoogle Scholar
  33. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365(6448):721–727. CrossRefPubMedGoogle Scholar
  34. Uchihashi T, Iino R, Ando T, Noji H (2011) High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333(6043):755–758. CrossRefPubMedGoogle Scholar
  35. Varley FS (1992) Neutron scattering lengths and cross section. Neutron News 3(3):26–37. CrossRefGoogle Scholar
  36. Wood RKS (1960) Pectic and cellulolytic enzymes in plant disease. Annu Rev Plant Physiol 11(1):299–322. CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Okazaki Institute for Integrative BioscienceNational Institutes of Natural SciencesAichiJapan
  2. 2.Department of Functional Molecular ScienceSchool of Physical SciencesKanagawaJapan
  3. 3.Institute for Molecular ScienceNational Institutes of Natural SciencesAichiJapan

Personalised recommendations