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Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I (Cel7A) Hydrolyzing Crystalline Cellulose Iα and IIII*

Open AccessPublished:April 01, 2014DOI:https://doi.org/10.1074/jbc.M113.546085
      Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.

      Introduction

      Enzyme activity on insoluble substrates is a common but poorly understood phenomenon in biological systems. The action of cellulolytic enzymes on cellulose is an important example of heterogeneous biocatalysis that has been the focus for ample research aimed at reducing the cost of lignocellulose-derived sugars for the production of biofuels (
      • Himmel M.E.
      • Ding S.Y.
      • Johnson D.K.
      • Adney W.S.
      • Nimlos M.R.
      • Brady J.W.
      • Foust T.D.
      Biomass recalcitrance: engineering plants and enzymes for biofuels production.
      ,
      • Chundawat S.P.
      • Beckham G.T.
      • Himmel M.E.
      • Dale B.E.
      Deconstruction of lignocellulosic biomass to fuels and chemicals.
      ,
      • Wilson D.B.
      Cellulases and biofuels.
      ,
      • Wilson D.B.
      Processive and nonprocessive cellulases for biofuel production: lessons from bacterial genomes and structural analysis.
      ). Fungal cellulases that hydrolyze crystalline cellulose share a common two-domain structure consisting of a catalytic domain and a cellulose binding domain, which promotes degradation by mediating adsorption of the cellulases on the cellulose surface (
      • Tomme P.
      • Van Tilbeurgh H.
      • Pettersson G.
      • Van Damme J.
      • Vandekerckhove J.
      • Knowles J.
      • Teeri T.
      • Claeyssens M.
      Studies of the cellulolytic system of Trichoderma reesei QM 9414: analysis of domain function in two cellobiohydrolases by limited proteolysis.
      ,
      • Reinikainen T.
      • Teleman O.
      • Teeri T.T.
      Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei.
      ). Recent studies using high speed atomic force microscopy (HS-AFM)
      The abbreviations used are:
      HS-AFM
      high speed atomic force microscope (microscopy)
      Cel7A
      cellobiohydrolase I
      Cy3-TrCel7A
      Cy3-labeled Trichoderma reesei cellobiohydrolase I
      pNPG
      p-nitrophenyl-β-d-glucoside
      pNPL
      p-nitrophenyl lactoside
      TrCel7A
      Trichoderma reesei cellobiohydrolase I
      TIRFM
      total internal reflection fluorescence microscope (microscopy).
      have revealed that cellobiohydrolase I (Cel7A) from Trichoderma reesei (TrCel7A) is a linear molecular motor, and productive binding to the reducing end of the crystalline cellulose results in translational movement to the nonreducing end (
      • Igarashi K.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Penttilä M.
      • Samejima M.
      High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose.
      ,
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ). However, the detailed mechanisms of action of TrCel7A, including kinetic constants of elementary reaction steps such as productive and nonproductive bindings, processive hydrolysis, and dissociation are not yet completely understood (Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Schematic model of the elementary reaction steps and kinetic parameters for crystalline cellulose hydrolysis by TrCel7A. In this scheme, TrCel7A first binds to the cellulose (binding rate constant kon). Then, nonproductively bound TrCel7A dissociates from cellulose surface without hydrolysis (nonproductive dissociation rate constant koffNP). Productively bound TrCel7A processively hydrolyzes glycosidic bonds of a cellulose chain along with translational movements (translational rate ktr), then dissociates from the cellulose surface (productive dissociation rate constant koffP).
      In addition to the elementary reaction steps of TrCel7A, it has been reported that the crystalline form of cellulose itself strongly affects hydrolysis by TrCel7A (
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase.
      ). Cellulose Iα and IIII are major commercial substrates that have received a lot of scientific and industrial attention lately. Treatment of cellulose Iα (naturally occurring crystalline cellulose) with supercritical ammonia results in conversion to cellulose IIII (
      • Wada M.
      • Heux L.
      • Isogai A.
      • Nishiyama Y.
      • Chanzy H.
      • Sugiyama J.
      Improved structural data of cellulose IIII prepared in supercritical ammonia.
      ). Crystalline cellulose Iα and IIII are allomorphs that differ in their hydrogen bonding network and stacking interaction patterns responsible for holding their cellulose chains together (
      • Wada M.
      • Heux L.
      • Isogai A.
      • Nishiyama Y.
      • Chanzy H.
      • Sugiyama J.
      Improved structural data of cellulose IIII prepared in supercritical ammonia.
      ,
      • Nishiyama Y.
      • Sugiyama J.
      • Chanzy H.
      • Langan P.
      Crystal structure and hydrogen bonding system in cellulose Iα from synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Chanzy H.
      • Nishiyama Y.
      • Langan P.
      Cellulose IIII crystal structure and hydrogen bonding by synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Nishiyama Y.
      • Langan P.
      X-ray structure of ammonia-cellulose I: new insights into the conversion of cellulose I to cellulose IIII.
      ). Recent works have reported that conversion of cellulose Iα into IIII enhances its enzymatic hydrolysis rates by up to 5-fold with micromolar enzyme concentrations (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ,
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase.
      ,
      • Chundawat S.P.
      • Bellesia G.
      • Uppugundla N.
      • da Costa Sousa L.
      • Gao D.
      • Cheh A.M.
      • Agarwal U.P.
      • Bianchetti C.M.
      • Phillips Jr., G.N.
      • Langan P.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate.
      ). Several studies have recently proposed that “decrystallization” of individual chains from the surface of crystalline cellulose is the key elementary step determining the susceptibility (
      • Beckham G.T.
      • Matthews J.F.
      • Peters B.
      • Bomble Y.J.
      • Himmel M.E.
      • Crowley M.F.
      Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.
      ,
      • Gao D.
      • Chundawat S.P.
      • Sethi A.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.
      ,
      • Payne C.M.
      • Jiang W.
      • Shirts M.R.
      • Himmel M.E.
      • Crowley M.F.
      • Beckham G.T.
      Glycoside hydrolase processivity is directly related to oligosaccharide binding free energy.
      ). Weaker intrasheet hydrogen bonding is considered to be one of the possible reasons for increased glucan chain flexibility and a lower thermodynamic barrier to individual chain extraction from the surface of cellulose IIII (
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase.
      ,
      • Chundawat S.P.
      • Bellesia G.
      • Uppugundla N.
      • da Costa Sousa L.
      • Gao D.
      • Cheh A.M.
      • Agarwal U.P.
      • Bianchetti C.M.
      • Phillips Jr., G.N.
      • Langan P.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate.
      ,
      • Beckham G.T.
      • Matthews J.F.
      • Peters B.
      • Bomble Y.J.
      • Himmel M.E.
      • Crowley M.F.
      Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.
      ,
      • Gao D.
      • Chundawat S.P.
      • Sethi A.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.
      ,
      • Payne C.M.
      • Jiang W.
      • Shirts M.R.
      • Himmel M.E.
      • Crowley M.F.
      • Beckham G.T.
      Glycoside hydrolase processivity is directly related to oligosaccharide binding free energy.
      ). However, the actual mechanism underlying the increased susceptibility of cellulose IIII remains unclear. With the HS-AFM, we have previously revealed that TrCel7A causes molecular congestion, or “traffic jams,” on the surface of crystalline cellulose (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ). Therefore, the traffic jams will also affect the susceptibility to hydrolysis by TrCel7A.
      In this study, we determined that susceptibility of cellulose Iα and IIII to hydrolysis by TrCel7A is highly dependent on enzyme concentration. Although cellulose IIII was more susceptible than cellulose Iα at micromolar enzyme concentration, no difference was observed at a nanomolar enzyme concentration. Furthermore, by means of single-molecule fluorescence imaging with total internal reflection fluorescence microscopy (TIRFM) and HS-AFM, we have directly visualized single TrCel7A molecules on cellulose Iα and IIII. The kinetic constants for elementary reaction steps of TrCel7A were determined and compared at picomolar enzyme concentration in which traffic jams do not occur on crystalline cellulose surfaces (Fig. 1). In all kinetic parameters measured, such as the binding rate constant, dissociation rate constant, and translational rate coupled with processive hydrolysis, large differences between cellulose Iα and IIII were not observed. Based on our results and the previously reported structural models of cellulose Iα and IIII (
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase.
      ,
      • Wada M.
      • Heux L.
      • Isogai A.
      • Nishiyama Y.
      • Chanzy H.
      • Sugiyama J.
      Improved structural data of cellulose IIII prepared in supercritical ammonia.
      ,
      • Nishiyama Y.
      • Sugiyama J.
      • Chanzy H.
      • Langan P.
      Crystal structure and hydrogen bonding system in cellulose Iα from synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Chanzy H.
      • Nishiyama Y.
      • Langan P.
      Cellulose IIII crystal structure and hydrogen bonding by synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Nishiyama Y.
      • Langan P.
      X-ray structure of ammonia-cellulose I: new insights into the conversion of cellulose I to cellulose IIII.
      ), we propose that different susceptibilities of cellulose Iα and IIII to hydrolysis by TrCel7A at high enzyme concentration arise from the surface properties of crystalline cellulose including ratio of hydrophobic surface and number of available lanes.

      DISCUSSION

      In the previous biochemical assay, cellulose Iα and IIII exhibit large differences regarding their susceptibility to hydrolysis by TrCel7A (
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase.
      ). However, in the present study, we found that susceptibility of cellulose Iα and IIII to TrCel7A is highly dependent on the enzyme concentration in the solution, and hydrolysis activities of TrCel7A against cellulose Iα and IIII are similar at low nanomolar concentrations (Table 1). With single-molecule imaging analysis, we also determined kinetic constants for elementary reaction steps of cellulose Iα and IIII hydrolysis by TrCel7A (Fig. 1). The results are summarized in Table 3. As suggested by the biochemical assay performed at nanomolar enzyme concentration, no significant differences were observed in all kinetic constants for TrCel7A against cellulose Iα and IIII.
      The binding modes of Cel7A to cellulose have been implicated as an important factor limiting efficient biocatalysis (
      • Chundawat S.P.
      • Beckham G.T.
      • Himmel M.E.
      • Dale B.E.
      Deconstruction of lignocellulosic biomass to fuels and chemicals.
      ,
      • Kurasin M.
      • Väljamäe P.
      Processivity of cellobiohydrolases is limited by the substrate.
      ,
      • Jalak J.
      • Väljamäe P.
      Mechanism of initial rapid rate retardation in cellobiohydrolase-catalyzed cellulose hydrolysis.
      ). The kon against single microfibril of cellulose IIII was only 1.5 times larger than that against cellulose Iα (Fig. 5A and Table 3). Note that the mean values of all kon for cellulose Iα and IIII were 1.6 × 109 ± 7.5 × 108 m−1μm−1s−1 (mean ± S.D., n = 64) and 2.3 × 109 ± 1.1 × 109 m−1μm−1s−1 (mean ± S.D., n = 169), respectively (Table 2), and the differences were <2-fold. Therefore, even if our attribution of the multiple peaks in kon distribution to the number of microfibrils in a bundle is not correct, the conclusion will not change. The values of kon for productive binding (or initial-cut product rate) have been reported in previous biochemical studies of Cel7A and crystalline cellulose from different sources (
      • Cruys-Bagger N.
      • Tatsumi H.
      • Ren G.R.
      • Borch K.
      • Westh P.
      Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain.
      ,
      • Fox J.M.
      • Levine S.E.
      • Clark D.S.
      • Blanch H.W.
      Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes.
      ), although the definition of kon in previous studies ((g/liter)−1s−1, g/liter corresponds to the substrate concentration) is different from ours (m−1μm−1s−1 or m−1μm−2s−1, m corresponds to the molar concentration of enzyme, Tables 2 and 3). The previously reported kon values are almost similar and ∼10−2 (g/liter)−1s−1 (
      • Cruys-Bagger N.
      • Tatsumi H.
      • Ren G.R.
      • Borch K.
      • Westh P.
      Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain.
      ,
      • Fox J.M.
      • Levine S.E.
      • Clark D.S.
      • Blanch H.W.
      Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes.
      ). The substrate concentrations used in the previous studies were ∼1 g/liter, which corresponds to the binding rate of ∼10−2 s−1 per reducing end. The previously reported kon values were obtained at 10−7–10−8 m enzyme concentrations, and the corresponding kon values in our study will be 102–103 μm−2s−1 (or 10−4–10−3 nm−2s−1) (Table 3). Considering the fraction of productive binding (∼0.5), area of β-glucose unit (∼0.2 nm2), and degree of polymerization of the cellulose (∼103) used in our study (
      • Yanagisawa M.
      • Shibata I.
      • Isogai A.
      SEC-MALLS analysis of softwood kraft pulp using LiCl/1,3-dimethyl-2-imidazolidinone as an eluent.
      ), this would correspond to the binding rate of 10−2–10−1 s−1 per reducing end. Thus, the kon values obtained in our study are similar or slightly larger compared with those previously reported.
      TrCel7A exhibited very similar koffP values on cellulose Iα and IIII in the present study (Fig. 6 and Table 3). These values were 10–103 times larger than those obtained by previous biochemical assays (
      • Kurasin M.
      • Väljamäe P.
      Processivity of cellobiohydrolases is limited by the substrate.
      ,
      • Cruys-Bagger N.
      • Tatsumi H.
      • Ren G.R.
      • Borch K.
      • Westh P.
      Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain.
      ). This can be attributed to the use of Cel7A and/or crystalline cellulose from different sources because our recent HS-AFM observation of TrCel7A on cellulose IIII obtained almost similar koffP (0.20 s−1) (
      • Nakamura A.
      • Watanabe H.
      • Ishida T.
      • Uchihashi T.
      • Wada M.
      • Ando T.
      • Igarashi K.
      • Samejima M.
      Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.
      ). According to the distribution of duration times on the cellulose surface, binding events of TrCel7A on both cellulose Iα and IIII included roughly half of productive binding that accompanies processive hydrolysis. Thus, there were no large differences in the binding modes of TrCel7A to cellulose Iα and IIII. As described above, degree of polymerization of the cellulose used in our study is ∼103 (
      • Yanagisawa M.
      • Shibata I.
      • Isogai A.
      SEC-MALLS analysis of softwood kraft pulp using LiCl/1,3-dimethyl-2-imidazolidinone as an eluent.
      ). Therefore, the density of the reducing end on the surface of crystalline cellulose will be 10−3 per β-glucose unit, and the fraction of TrCel7A that finds the reducing end will be 10−3 when TrCel7A binds randomly to the cellulose surface. This implies that TrCel7A binds to the reducing end with remarkable specificity.
      The ktr values for TrCel7A were almost the same when comparing cellulose Iα and IIII (Fig. 7). Considering the length of a cellobiose unit (∼1 nm), the ktr represents not only the translational rate but also turnover rate of TrCel7A against cellulose Iα and IIII and can be estimated as 5.0 s−1 and 5.1 s−1, respectively. Furthermore, the values of koffP correspond to the time constants of 8.3 s and 7.1 s for cellulose Iα and IIII, respectively. Therefore, the expected values of processivity for Cy3-TrCel7A on cellulose Iα and IIII are estimated to be 42 (5.0 s−1 × 8.3 s) and 36 (5.1 s−1 × 7.1 s), respectively. These values are comparable with those of TrCel7A on bacterial cellulose previously determined biochemically (
      • Jalak J.
      • Kurašin M.
      • Teugjas H.
      • Väljamäe P.
      Endo-exo synergism in cellulose hydrolysis revisited.
      ), and on cellulose IIII obtained by our recent HS-AFM observation (
      • Nakamura A.
      • Watanabe H.
      • Ishida T.
      • Uchihashi T.
      • Wada M.
      • Ando T.
      • Igarashi K.
      • Samejima M.
      Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.
      ). The good accordance of processivity supports our interpretation that the long component of koff corresponds to the productive binding.
      Our results indicate that the effect of conversion of cellulose Iα into IIII on the susceptibility at high enzyme concentration does not come from the different kon, ktr, and koff. One possible factor determining the susceptibility is the decrystallization step as previously proposed (
      • Beckham G.T.
      • Matthews J.F.
      • Peters B.
      • Bomble Y.J.
      • Himmel M.E.
      • Crowley M.F.
      Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.
      ,
      • Gao D.
      • Chundawat S.P.
      • Sethi A.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.
      ,
      • Payne C.M.
      • Jiang W.
      • Shirts M.R.
      • Himmel M.E.
      • Crowley M.F.
      • Beckham G.T.
      Glycoside hydrolase processivity is directly related to oligosaccharide binding free energy.
      ). However, dependence of cellulose Iα susceptibility on TrCel7A concentration is not apparently consistent with the notion that decrystallization is a rate-limiting step (Table 1). If the decrystallization is slow on cellulose Iα, a transient pause before translational movement will occur after initial binding to the reducing end. Although we could not resolve decrystallization step from translational movement in our single-molecule fluorescence imaging, the decrystallization step may be directly visualized with high resolution HS-AFM observation. It will be an interesting issue to be investigated in the future study.
      In addition to the decrystallization as an elementary reaction step (
      • Igarashi K.
      • Wada M.
      • Samejima M.
      Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase.
      ,
      • Chundawat S.P.
      • Bellesia G.
      • Uppugundla N.
      • da Costa Sousa L.
      • Gao D.
      • Cheh A.M.
      • Agarwal U.P.
      • Bianchetti C.M.
      • Phillips Jr., G.N.
      • Langan P.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate.
      ,
      • Beckham G.T.
      • Matthews J.F.
      • Peters B.
      • Bomble Y.J.
      • Himmel M.E.
      • Crowley M.F.
      Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.
      ,
      • Gao D.
      • Chundawat S.P.
      • Sethi A.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.
      ,
      • Payne C.M.
      • Jiang W.
      • Shirts M.R.
      • Himmel M.E.
      • Crowley M.F.
      • Beckham G.T.
      Glycoside hydrolase processivity is directly related to oligosaccharide binding free energy.
      ), the force generated by TrCel7A in the decrystallization step will be another interesting issue to be resolved. To understand chemo-mechanical coupling and force generation mechanisms of TrCel7A as a molecular motor, visualization of the steps and pauses for moving TrCel7A is indispensable (
      • Bustamante C.
      • Chemla Y.R.
      • Forde N.R.
      • Izhaky D.
      Mechanical processes in biochemistry.
      ). This will require single-molecule measurements with subnanometer and microsecond spatio-temporal resolution (
      • Greenleaf W.J.
      • Woodside M.T.
      • Block S.M.
      High-resolution, single-molecule measurements of biomolecular motion.
      ). Although HS-AFM is a powerful technique that can directly visualize movements and conformational changes of molecular motors with high spatial resolution (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ,
      • Uchihashi T.
      • Iino R.
      • Ando T.
      • Noji H.
      High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase.
      ), temporal resolution is limited to several tens of milliseconds (
      • Ando T.
      • Uchihashi T.
      • Kodera N.
      High-speed AFM and applications to biomolecular systems.
      ). Single-molecule analysis of TrCel7A with high spatio-temporal resolution with optical microscopy can be achieved with larger probes such as colloidal gold, which enables high image contrast (
      • Ueno H.
      • Nishikawa S.
      • Iino R.
      • Tabata K.V.
      • Sakakihara S.
      • Yanagida T.
      • Noji H.
      Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.
      ,
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ).
      The diameters of cellulose Iα and IIII microfibrils used in our study were almost the same (Fig. 5C). However, it has been reported that cellulose Iα and IIII have different crystalline forms (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ,
      • Nishiyama Y.
      • Sugiyama J.
      • Chanzy H.
      • Langan P.
      Crystal structure and hydrogen bonding system in cellulose Iα from synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Chanzy H.
      • Nishiyama Y.
      • Langan P.
      Cellulose IIII crystal structure and hydrogen bonding by synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Nishiyama Y.
      • Langan P.
      X-ray structure of ammonia-cellulose I: new insights into the conversion of cellulose I to cellulose IIII.
      ). Cellulose Iα has narrow hydrophobic 110 surfaces between wide hydrophilic 100 and 010 surfaces, whereas cellulose IIII has wide moderately hydrophobic 100 surfaces in addition to narrow hydrophobic 1̄10 surface and wide hydrophilic 010 surface (Fig. 8A) (
      • Igarashi K.
      • Uchihashi T.
      • Koivula A.
      • Wada M.
      • Kimura S.
      • Okamoto T.
      • Penttilä M.
      • Ando T.
      • Samejima M.
      Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.
      ,
      • Nishiyama Y.
      • Sugiyama J.
      • Chanzy H.
      • Langan P.
      Crystal structure and hydrogen bonding system in cellulose Iα from synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Chanzy H.
      • Nishiyama Y.
      • Langan P.
      Cellulose IIII crystal structure and hydrogen bonding by synchrotron x-ray and neutron fiber diffraction.
      ,
      • Wada M.
      • Nishiyama Y.
      • Langan P.
      X-ray structure of ammonia-cellulose I: new insights into the conversion of cellulose I to cellulose IIII.
      ,
      • Chundawat S.P.
      • Bellesia G.
      • Uppugundla N.
      • da Costa Sousa L.
      • Gao D.
      • Cheh A.M.
      • Agarwal U.P.
      • Bianchetti C.M.
      • Phillips Jr., G.N.
      • Langan P.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate.
      ). Considering that TrCel7A primarily binds to hydrophobic surfaces, a large moderately hydrophobic surface in cellulose IIII will increase number of accessible reducing ends in single microfibril. Therefore, actual kon for the single reducing end may be different between cellulose Iα and IIII as proposed in previous study (
      • Gao D.
      • Chundawat S.P.
      • Sethi A.
      • Balan V.
      • Gnanakaran S.
      • Dale B.E.
      Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis.
      ). It is also highly probable that cellulose IIII has an increased number of available lanes for TrCel7A movement relative to cellulose Iα. This would relieve the traffic jams of TrCel7A on cellulose IIII. However, quantitative comparison of the degree of traffic jams between cellulose Iα and IIII at defined enzyme concentration was difficult due to strong and significant nonspecific bindings of TrCel7A to the graphite used as a surface to which cellulose Iα and IIII were immobilized in HS-AFM observation.
      Figure thumbnail gr8
      FIGURE 8Model of different susceptibilities of cellulose Iα and IIII to hydrolysis by TrCel7A. A, schematics show cross-sections of cellulose Iα (left) and IIII (right). B, compared with cellulose Iα (left), cellulose IIII (right) has a largely increased number of accessible reducing ends for TrCel7A binding and lanes for TrCel7A translational movement. At low TrCel7A concentration (picomolar to nanomolar) (top), accessible reducing ends and lanes are sufficient on both cellulose Iα and IIII. At high TrCel7A concentration (micromolar) (bottom), shortage of reducing ends and/or traffic jams occurs on cellulose Iα, but not on cellulose IIII.
      Finally, we propose our current model of different susceptibilities of cellulose Iα and IIII to hydrolysis by TrCel7A. At low TrCel7A concentration (picomolar to nanomolar) (Fig. 8B, top), accessible reducing ends for TrCel7A binding and lanes for TrCel7A translational movement are sufficient on both cellulose Iα and IIII, and both cellulose Iα and IIII are susceptible. At high concentration (micromolar), however, TrCel7A will easily cause shortage in available reducing ends and/or traffic jams on cellulose Iα but not on cellulose IIII, which results in low susceptibility of cellulose Iα (Fig. 8B). Therefore, optimization of enzyme concentration and hydrophobicity of the crystalline cellulose surface will be essential for efficient industrial production of cellulosic biofuels with TrCel7A.

      Acknowledgments

      We thank Prof. Masahisa Wada (The University of Tokyo) for technical help in preparing crystalline celluloses and the members of the laboratory for valuable comments.

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