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Masahiro Samejima - One of the best experts on this subject based on the ideXlab platform.
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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, 2016Co-Authors: Akihiko Nakamura, Hiroyuki Noji, Tomoyuki Tasaki, Daiki Ishiwata, Mayuko Yamamoto, Yasuko Okuni, Akasit Visootsat, Morice Maximilien, Taku Uchiyama, Masahiro SamejimaAbstract: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 Iα. 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.
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single molecule imaging analysis of elementary reaction steps of trichoderma reesei cellobiohydrolase i cel7a hydrolyzing Crystalline Cellulose iα and iiii
Journal of Biological Chemistry, 2014Co-Authors: Yusuke Shibafuji, Akihiko Nakamura, Hiroki Watanabe, Takayuki Uchihashi, Toshio Ando, Masahiro Samejima, Naohisa Sugimoto, Shingo Fukuda, Hiroyuki Noji, Anu KoivulaAbstract:Abstract 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 owing to 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.
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single molecule imaging analysis of elementary reaction steps of trichoderma reesei cellobiohydrolase i cel7a hydrolyzing Crystalline Cellulose iα and iiii
Journal of Biological Chemistry, 2014Co-Authors: Yusuke Shibafuji, Akihiko Nakamura, Hiroki Watanabe, Takayuki Uchihashi, Toshio Ando, Masahiro Samejima, Naohisa Sugimoto, Shingo Fukuda, Hiroyuki Noji, Anu KoivulaAbstract: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.
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the tryptophan residue at the active site tunnel entrance of trichoderma reesei cellobiohydrolase cel7a is important for initiation of degradation of Crystalline Cellulose
Journal of Biological Chemistry, 2013Co-Authors: Akihiko Nakamura, Masahisa Wada, Kiyohiko Igarashi, Anu Koivula, Takeshi Tsukada, Sanna Auer, Tadaomi Furuta, Masahiro SamejimaAbstract:The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly Crystalline Cellulose. The catalytic domain and the Cellulose-binding domain (CBD) are both necessary for full activity on Crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade Crystalline Cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on Crystalline Cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite −7. However, when similar simulations were carried out starting from subsite −5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between Crystalline and amorphous Celluloses. The CBD is important for enhancing the enzyme population on Crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the Cellulose surface and/or hindering processive hydrolysis.
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adsorption characteristics of fungal family 1 Cellulose binding domain from trichoderma reesei cellobiohydrolase i on Crystalline Cellulose negative cooperative adsorption via a steric exclusion effect
Langmuir, 2012Co-Authors: Naohisa Sugimoto, Masahisa Wada, Kiyohiko Igarashi, Masahiro SamejimaAbstract:Cellobiohydrolases (CBHs) hydrolyzing Crystalline Cellulose share a two-domain structure of catalytic domain (CD) and Cellulose-binding domain (CBD). To focus on the binding characteristics of CBD,...
Masahisa Wada - One of the best experts on this subject based on the ideXlab platform.
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trade off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of Crystalline Cellulose
Journal of the American Chemical Society, 2014Co-Authors: Akihiko Nakamura, Hiroki Watanabe, Takuya Ishida, Takayuki Uchihashi, Masahisa Wada, Toshio AndoAbstract:Analysis of heterogeneous catalysis at an interface is difficult because of the variety of reaction sites and the difficulty of observing the reaction. Enzymatic hydrolysis of Cellulose by cellulases is a typical heterogeneous reaction at a solid/liquid interface, and a key parameter of such reactions on polymeric substrates is the processivity, i.e., the number of catalytic cycles that can occur without detachment of the enzyme from the substrate. In this study, we evaluated the reactions of three closely related glycoside hydrolase family 7 cellobiohydrolases from filamentous fungi at the molecular level by means of high-speed atomic force microscopy to investigate the structure–function relationship of the cellobiohydrolases on Crystalline Cellulose. We found that high moving velocity of enzyme molecules on the surface is associated with a high dissociation rate constant from the substrate, which means weak interaction between enzyme and substrate. Moreover, higher values of processivity were associate...
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the tryptophan residue at the active site tunnel entrance of trichoderma reesei cellobiohydrolase cel7a is important for initiation of degradation of Crystalline Cellulose
Journal of Biological Chemistry, 2013Co-Authors: Akihiko Nakamura, Masahisa Wada, Kiyohiko Igarashi, Anu Koivula, Takeshi Tsukada, Sanna Auer, Tadaomi Furuta, Masahiro SamejimaAbstract:The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly Crystalline Cellulose. The catalytic domain and the Cellulose-binding domain (CBD) are both necessary for full activity on Crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade Crystalline Cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on Crystalline Cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite −7. However, when similar simulations were carried out starting from subsite −5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between Crystalline and amorphous Celluloses. The CBD is important for enhancing the enzyme population on Crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the Cellulose surface and/or hindering processive hydrolysis.
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adsorption characteristics of fungal family 1 Cellulose binding domain from trichoderma reesei cellobiohydrolase i on Crystalline Cellulose negative cooperative adsorption via a steric exclusion effect
Langmuir, 2012Co-Authors: Naohisa Sugimoto, Masahisa Wada, Kiyohiko Igarashi, Masahiro SamejimaAbstract:Cellobiohydrolases (CBHs) hydrolyzing Crystalline Cellulose share a two-domain structure of catalytic domain (CD) and Cellulose-binding domain (CBD). To focus on the binding characteristics of CBD,...
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visualization of cellobiohydrolase i from trichoderma reesei moving on Crystalline Cellulose using high speed atomic force microscopy
Methods in Enzymology, 2012Co-Authors: Kiyohiko Igarashi, Takayuki Uchihashi, Masahisa Wada, Toshio Ando, Anu Koivula, Satoshi Kimura, Merja Penttila, Masahiro SamejimaAbstract:Cellulases hydrolyze β-1,4-glucosidic linkages of insoluble Cellulose at the solid/liquid interface, generating soluble cellooligosaccharides. We describe here our method for real-time observation of the behavior of cellulase molecules on the substrate, using high-speed atomic force microscopy (HS-AFM). When glycoside hydrolase family 7 cellobiohydrolase from Trichoderma reesei (TrCel7A) was incubated with Crystalline Cellulose, many enzyme molecules were observed to move unidirectionally on the surface of the substrate by HS-AFM. The velocity of the moving molecules of TrCel7A on Cellulose I crystals was estimated by means of image analysis.
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traffic jams reduce hydrolytic efficiency of cellulase on Cellulose surface
Science, 2011Co-Authors: Kiyohiko Igarashi, Takayuki Uchihashi, Masahisa Wada, Anu Koivula, Satoshi Kimura, Tetsuaki Okamoto, Merja PenttilaAbstract:A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of Crystalline Cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the Crystalline Cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the Crystalline polymorphic form of Cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the Crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky Crystalline Cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.
Anu Koivula - One of the best experts on this subject based on the ideXlab platform.
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single molecule imaging analysis of elementary reaction steps of trichoderma reesei cellobiohydrolase i cel7a hydrolyzing Crystalline Cellulose iα and iiii
Journal of Biological Chemistry, 2014Co-Authors: Yusuke Shibafuji, Akihiko Nakamura, Hiroki Watanabe, Takayuki Uchihashi, Toshio Ando, Masahiro Samejima, Naohisa Sugimoto, Shingo Fukuda, Hiroyuki Noji, Anu KoivulaAbstract:Abstract 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 owing to 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.
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single molecule imaging analysis of elementary reaction steps of trichoderma reesei cellobiohydrolase i cel7a hydrolyzing Crystalline Cellulose iα and iiii
Journal of Biological Chemistry, 2014Co-Authors: Yusuke Shibafuji, Akihiko Nakamura, Hiroki Watanabe, Takayuki Uchihashi, Toshio Ando, Masahiro Samejima, Naohisa Sugimoto, Shingo Fukuda, Hiroyuki Noji, Anu KoivulaAbstract: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.
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the tryptophan residue at the active site tunnel entrance of trichoderma reesei cellobiohydrolase cel7a is important for initiation of degradation of Crystalline Cellulose
Journal of Biological Chemistry, 2013Co-Authors: Akihiko Nakamura, Masahisa Wada, Kiyohiko Igarashi, Anu Koivula, Takeshi Tsukada, Sanna Auer, Tadaomi Furuta, Masahiro SamejimaAbstract:The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly Crystalline Cellulose. The catalytic domain and the Cellulose-binding domain (CBD) are both necessary for full activity on Crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade Crystalline Cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on Crystalline Cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite −7. However, when similar simulations were carried out starting from subsite −5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between Crystalline and amorphous Celluloses. The CBD is important for enhancing the enzyme population on Crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the Cellulose surface and/or hindering processive hydrolysis.
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visualization of cellobiohydrolase i from trichoderma reesei moving on Crystalline Cellulose using high speed atomic force microscopy
Methods in Enzymology, 2012Co-Authors: Kiyohiko Igarashi, Takayuki Uchihashi, Masahisa Wada, Toshio Ando, Anu Koivula, Satoshi Kimura, Merja Penttila, Masahiro SamejimaAbstract:Cellulases hydrolyze β-1,4-glucosidic linkages of insoluble Cellulose at the solid/liquid interface, generating soluble cellooligosaccharides. We describe here our method for real-time observation of the behavior of cellulase molecules on the substrate, using high-speed atomic force microscopy (HS-AFM). When glycoside hydrolase family 7 cellobiohydrolase from Trichoderma reesei (TrCel7A) was incubated with Crystalline Cellulose, many enzyme molecules were observed to move unidirectionally on the surface of the substrate by HS-AFM. The velocity of the moving molecules of TrCel7A on Cellulose I crystals was estimated by means of image analysis.
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traffic jams reduce hydrolytic efficiency of cellulase on Cellulose surface
Science, 2011Co-Authors: Kiyohiko Igarashi, Takayuki Uchihashi, Masahisa Wada, Anu Koivula, Satoshi Kimura, Tetsuaki Okamoto, Merja PenttilaAbstract:A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of Crystalline Cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the Crystalline Cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the Crystalline polymorphic form of Cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the Crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky Crystalline Cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.
Kiyohiko Igarashi - One of the best experts on this subject based on the ideXlab platform.
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the tryptophan residue at the active site tunnel entrance of trichoderma reesei cellobiohydrolase cel7a is important for initiation of degradation of Crystalline Cellulose
Journal of Biological Chemistry, 2013Co-Authors: Akihiko Nakamura, Masahisa Wada, Kiyohiko Igarashi, Anu Koivula, Takeshi Tsukada, Sanna Auer, Tadaomi Furuta, Masahiro SamejimaAbstract:The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly Crystalline Cellulose. The catalytic domain and the Cellulose-binding domain (CBD) are both necessary for full activity on Crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade Crystalline Cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on Crystalline Cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite −7. However, when similar simulations were carried out starting from subsite −5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between Crystalline and amorphous Celluloses. The CBD is important for enhancing the enzyme population on Crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the Cellulose surface and/or hindering processive hydrolysis.
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adsorption characteristics of fungal family 1 Cellulose binding domain from trichoderma reesei cellobiohydrolase i on Crystalline Cellulose negative cooperative adsorption via a steric exclusion effect
Langmuir, 2012Co-Authors: Naohisa Sugimoto, Masahisa Wada, Kiyohiko Igarashi, Masahiro SamejimaAbstract:Cellobiohydrolases (CBHs) hydrolyzing Crystalline Cellulose share a two-domain structure of catalytic domain (CD) and Cellulose-binding domain (CBD). To focus on the binding characteristics of CBD,...
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visualization of cellobiohydrolase i from trichoderma reesei moving on Crystalline Cellulose using high speed atomic force microscopy
Methods in Enzymology, 2012Co-Authors: Kiyohiko Igarashi, Takayuki Uchihashi, Masahisa Wada, Toshio Ando, Anu Koivula, Satoshi Kimura, Merja Penttila, Masahiro SamejimaAbstract:Cellulases hydrolyze β-1,4-glucosidic linkages of insoluble Cellulose at the solid/liquid interface, generating soluble cellooligosaccharides. We describe here our method for real-time observation of the behavior of cellulase molecules on the substrate, using high-speed atomic force microscopy (HS-AFM). When glycoside hydrolase family 7 cellobiohydrolase from Trichoderma reesei (TrCel7A) was incubated with Crystalline Cellulose, many enzyme molecules were observed to move unidirectionally on the surface of the substrate by HS-AFM. The velocity of the moving molecules of TrCel7A on Cellulose I crystals was estimated by means of image analysis.
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traffic jams reduce hydrolytic efficiency of cellulase on Cellulose surface
Science, 2011Co-Authors: Kiyohiko Igarashi, Takayuki Uchihashi, Masahisa Wada, Anu Koivula, Satoshi Kimura, Tetsuaki Okamoto, Merja PenttilaAbstract:A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of Crystalline Cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the Crystalline Cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the Crystalline polymorphic form of Cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the Crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky Crystalline Cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.
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high speed atomic force microscopy visualizes processive movement of trichoderma reesei cellobiohydrolase i on Crystalline Cellulose
Journal of Biological Chemistry, 2009Co-Authors: Kiyohiko Igarashi, Masahisa Wada, Anu Koivula, Satoshi Kimura, Merja Penttila, Masahiro SamejimaAbstract: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.
Thomas M. Wood - One of the best experts on this subject based on the ideXlab platform.
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the cellulase system of the anaerobic rumen fungus neocallimastix frontalis studies on the properties of fractions rich in endo 1 4 β d glucanase activity
Applied Microbiology and Biotechnology, 1995Co-Authors: Thomas M. Wood, Catriona A Wilson, Sheila I. MccraeAbstract:Seven fractions rich in endoglucanase activity were separated from the extracellular cellulase system of the anaerobic rumen fungus Neocallimastix frontalis. The fractions (ES1, ES3, ES2U1, ES2U2, ES2U4, ES2U3C1 and ES2U3C2) were separated from each other and from a fraction that could solubilize Crystalline Cellulose (the so-called Crystalline-Cellulose-solubilizing component, CCSC) by the sequential use of differential adsorption on the microCrystalline Cellulose Avicel, gel filtration and affinity chromatography on concanavalin-A–Sepharose. The molecular masses of the endoglucanase fractions, when determined by gel filtration, were 64, 30, 61, 113, 17, 38 and 93 kDa respectively. Each enzyme degraded carboxymethylCellulose and was rich in activity to Cellulose swollen in phosphoric acid to break the hydrogen bonding: cellobiose, cellotriose and cellotetraose were released in differing proportions. Each fraction showed a characteristic gradient when the capacity of each enzyme to increase the fluidity of a solution of carboxymethylCellulose was plotted against the increase in reducing power of the solution. Although neither endoglucanase fraction, acting in isolation, could degrade Crystalline Cellulose, three of the fractions (ES1, ES3 and ES2U1) could act synergistically with the CCSC fraction in this regard. Remarkably, the same three fractions also acted in synergism with the cellobiohydrolases (CBH I and CBH II) of the aerobic fungus Penicillium pinophilum in degrading Crystalline Cellulose, but only when both cellobiohydrolase enzymes were present in the solution along with any one of the three endoglucanases. These observations support the conclusion that the mechanism of action of the cellulase system of N. frontalis in degrading Crystalline Cellulose may be similar to that operating in the aerobic fungi.
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synergism between components of the cellulase system of the anaerobic rumen fungus neocallimastix frontalis and those of the aerobic fungi penicillium pinophilum and trichoderma koningii in degrading Crystalline Cellulose
Applied Microbiology and Biotechnology, 1994Co-Authors: Thomas M. Wood, Catriona A Wilson, Sheila I. MccraeAbstract:The cellulase system of Neocallimastix frontalis was separated by differential affinity on Cellulose into an adsorbed fraction that could solubilize Crystalline Cellulose (Crystalline-Cellulose-solubilizing fraction, CCSF), and a non-adsorbed fraction that contained endoglucanase and β-glucosidase activities (non-adsorbed endoglucanase/ β-glucosidas, NAE/β-G) but which showed no activity to Crystalline Cellulose. Both fractions were tested for their capacity to act synergistically with the cellobiohydrolase (CBH) components of aerobic fungi in degrading Crystalline Cellulose. The CCSF acted synergistically with CBH I components of both Penicillium pinophilum and Trichoderma koningii but not with CBH II. The NAE/β-G fraction also acted synergistically with the CBH components of P. pinophilum but, remarkably, only when both CBH I and CBH II were present in the reaction mixture. By comparison with previously published studies on the mechanism of action of P. pinophilum cellulase it is speculated that the CCSF of N. frontalis may contain CBH I- and CBH II-type enzymes.
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studies on the cellulase of the rumen anaerobic fungus neocallimastix frontalis with special reference to the capacity of the enzyme to degrade Crystalline Cellulose
Enzyme and Microbial Technology, 1992Co-Authors: Catriona A Wilson, Thomas M. WoodAbstract:Abstract By using cotton fiber, carboxymethylCellulose (CM-Cellulose), and o -nitrophenyl-β- d -glucoside as substrates, it was possible to demonstrate that there were at least three different types of enzyme present in culture filtrates of Neocallimastix frontalis RK21. The activity to Crystalline Cellulose (cotton fiber) resided in a high-molecular-weight complex that comprised endoglucanase activity, β-glucosidase activity, and another enzyme. However, synergism between the components in the high-molecular-weight complex and between the complex and low-molecular-weight endoglucanases and β-glucosidases was also apparent in the solubilization of Crystalline Cellulose. The composition of the complex varied according to the growth conditions: it was, however, between 750 and 1000 kDa in size. Cultures containing rumen fluid contained only small amounts of the high-molecular-weight complex. Cultures grown on defined medium were rich in high-molecular-weight complex, but only when the concentration of the carbon source was less than 1.0%. Treatment of the crude culture filtrates with chitinase under conditions that had little effect on the activity of the enzyme to endoglucanase (CM-Cellulose) or β-glucosidase completely destroyed the activity to Crystalline Cellulose. It is tentatively suggested that an enzyme crucial for the activity to Crystalline Cellulose may be cell wall-bound and may be dependent on its association with the cell wall for the maintenance of its conformation for attacking Crystalline Cellulose. The enzyme involved in degrading Crystalline Cellulose is much more thermolabile than the CM-cellulase or the β-glucosidase: activity is optimal at pH 6.0 and 40°C.