High Speed Atomic Force Microscopy Visualizes Processive Movement of Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose

Abstract

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.

Trade-off between Processivity and Hydrolytic Velocity of Cellobiohydrolases at the Surface of Crystalline Cellulose

“…The velocity of processive movement estimated from the HS-AFM observation and the velocities of cellobiose production determined by biochemical experiments were very different as reported previously 4,34 (Table 1). Considering only reactive (working) enzyme molecules are visualized in the HS-AFM observation and biochemical bulk experiments cause underestimation of the specific activity of the CBHs, the number of active enzymes was quite limited (<1% of added enzyme) at the surface of the crystalline cellulose.…”

High-speed atomic force microscopy coming of age

“…Imaging rate, 1.03 fps (×20 playback); scan size, 800 × 800 nm 2 [98]. ・Crystallization of a CaCO 3 thin film from supersaturated solution [72] ・Ultrafast imaging of collagen [58] ・Brownian motion & photo-degradation of π-conjugated polyrotaxane [73] ・DNA translocation and looping by type III restriction enzyme [74] ・Biotinylated DNA-streptavidin interaction [75] Miles Miles Shinohara Takeyasu Trimitsu 2008 5 ・Anisotropic diffusion of point defects in streptavidin 2D crystals [76] ・Identification of intrinsically disordered regions of proteins [77] ・Human chromosomes in liquid [78] ・DNA-nuclease interaction [ ・Dynamic equilibrium at the edge of bR 2D crystals [81] ・Structural change of CaM and actin polymerization on streptavidin 2D crystals [82] ・Unidirectional translocation of cellulase along cellulose fibers [83] ・Translocation of EcoRII restriction enzyme along DNA [84] ・Fabrication and imaging of hard material surface [85] ・Purple membrane in contact-mode HS-AFM [86] ・Thermal motion of π-conjugated polymer chain [87] ・Opening of 3D hollow structure of DNA Origami [88] ・Opening of 3D hollow structure of DNA Origami [89] ・ATP-induced conformational change in P2X 4 ・Walking myosin V along an actin filament [91] ・Photo-induced structural change in bR [92] ・2D crystal formation of annexin A-V and height change of p97 [93] ・Analysis of components covering magnesotome surface [94] ・Time course of cell death by antimicrobial peptide [95] ・Dissolution of extreme UV exposed resist films under developing [96] ・Dissolution of extreme UV exposed resist films under developing [97] ・Process of forming supported planar lipid bilayer [98] ・Self assembly of amyloid-like fibrils [99] ・Effect of ClpX on FtsZ polymerization ...…”

“…From biochemical kinetics analyses, TrCel7A is thought to consecutively hydrolyze the crystalline cellulose chain without releasing the chain, and therefore, is expected to processively move in one direction during the hydrolysis reaction [170]. Nevertheless, this long-standing issue had never been directly revealed before the high-speed AFM observation recently conducted by Kiyohiko Igarashi and collaborators [83] and by the collaboration between my group and Kiyohiko Igarashi's group [115].…”

“…Typical examples of this class are the studies on unidirectional translocation of cellulase along cellulose fibers [83,115] and transportation of synthetic DNA motor along a series of DNA stators [118]. The study on translocation of EcoRII restriction enzyme along DNA [74] also belongs to this class.…”

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

“…Since the molecular chains of chitin are packed with parallel orientation in the crystal 7 , the processive movement of the enzyme molecules can be directly visualized using the procedure described in our previous reports on HS-AFM studies of processive cellulases from the cellulolytic ascomycete Trichoderma reesei [8][9][10] . There was an advantage of using chitinases rather than cellulases for the present work: the chitinolytic bacterium S. marcescens 11 produces only two extracellular processive chitinases, ChiA and ChiB, and these two enzymes comprise one of the best studied pairs of processive enzymes acting on cellulose/chitin 12 .…”