High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose.
Fungal cellobiohydrolases act at liquid-solid interfaces. They have the ability to hydrolyze cellulose chains of a crystalline substrate because of their two-domain structure, i.e. cellulose-binding domain and catalytic domain, and unique active site architecture. However, the details of the action of the two domains on crystalline cellulose are still unclear. Here, we present real time observations of Trichoderma reesei (Tr) cellobiohydrolase I (Cel7A) molecules sliding on crystalline cellulose, obtained with a high speed atomic force microscope. The average velocity of the sliding movement on crystalline cellulose was 3.5 nm/s, and interestingly, the catalytic domain without the cellulose-binding domain moved with a velocity similar to that of the intact TrCel7A enzyme. However, no sliding of a catalytically inactive enzyme (mutant E212Q) or a variant lacking tryptophan at the entrance of the active site tunnel (mutant W40A) could be detected. This indicates that, besides the hydrolysis of glycosidic bonds, the loading of a cellulose chain into the active site tunnel is also essential for the enzyme movement.
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Electronic address: aquarius@mail.ecc.u-tokyo.ac.jp.
Kiyohiko
K
Igarashi
VTT Technical Research Centre of Finland, Espoo, 02044 VTT, Finland.
Anu
A
Koivula
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
Masahisa
M
Wada
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
Satoshi
S
Kimura
VTT Technical Research Centre of Finland, Espoo, 02044 VTT, Finland.
Merja
M
Penttilä
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
Masahiro
M
Samejima
D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000502:physiology; D002482:Cellulose / Q000737:chemistry* / Q000648:ultrastructure; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000502:physiology; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase