The Tryptophan Residue at the Active Site Tunnel Entrance of Trichoderma reesei Cellobiohydrolase Cel7A Is Important for Initiation of Degradation of Crystalline Cellulose

Nakamura, Akihiko; Tsukada, Takeshi; Auer, Sanna; Furuta, Tadaomi; Wada, Masahisa; Koivula, Anu; Igarashi, Kiyohiko; Samejima, Masahiro

doi:10.1074/jbc.m113.452623

Cited by 84 publications

(84 citation statements)

References 36 publications

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“…40 In addition, because PASC contains large numbers of cellulose chain ends and free chains for the initiation of endotype hydrolysis, 32 capturing the substrate chain by CD or decrystallization of the substrate is not a rate-limiting step. 41 In the hydrolysis of crystalline cellulose I α , the relationship among the three enzymes was similar to that of processivity whereas the difference between PcCel7D and TrCel7A was smaller in cellulose III I hydrolysis. The big difference between the two crystalline celluloses is the shape of the crystal.…”

Section: ■ Discussionmentioning

confidence: 88%

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

et al. 2014

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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 associated with more loop regions covering the subsite cleft, which may imply higher binding affinity. Loop regions covering the subsites result in stronger interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-off between processivity and hydrolytic velocity among processive cellulases.

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Section: ■ Discussionmentioning

confidence: 88%

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

et al. 2014

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“…One suggested role of Trp is that "stacking" interactions of -electrons in its aromatic ring and the ␣-face of the carbohydrate (50) enable the necessary sliding of the bound chain (4, 51), possibly through stabilization of the transition state between adjacent positions in the processive movement (52,53). Tryptophan residues near the entrance of the active tunnel are also important for the initial "threading" and recognition of the cellulose strand, particularly on crystalline substrates (16,26,38,44,54). In the current work, we used the W38A mutation in T. reesei Cel7A to obtain an enzyme with weakened substrate affinity.…”

Section: Discussionmentioning

confidence: 99%

“…Many mutational studies in which Trp has been replaced with nonaromatic residues have shown negative effects on activity and processivity of, for example, cellulases (16,36,38,(43)(44)(45)(46) and chitinases (47)(48)(49). One suggested role of Trp is that "stacking" interactions of -electrons in its aromatic ring and the ␣-face of the carbohydrate (50) enable the necessary sliding of the bound chain (4, 51), possibly through stabilization of the transition state between adjacent positions in the processive movement (52,53).…”

Section: Discussionmentioning

confidence: 99%

“…Numerous earlier studies have addressed this question, and interestingly, it appears as if the conclusions fall mainly into two mutually conflicting groups. Thus, a number of works have used either direct experimental evidence or re-examination of broader, previously published material to conclude that the overall rate is governed by the association of enzyme and substrate, particularly the placement of a cellulose strand in the long active tunnel (11)(12)(13)(14)(15)(16)(17)(18). In contrast to this, other studies, including work by this group on the pre-steady state kinetics of wild type Cel7A, have suggested that the formation of the activated complex is quite rapid and that dissociation of enzyme, which is in a position where further processive movement is hindered, is the rate-limiting step (19 -25).…”

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Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

Kari

Olsen

Borch

et al. 2014

Journal of Biological Chemistry

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Background:To elucidate the rate-determining steps of cellobiohydrolase Cel7A from T. reesei, variants with lower substrate affinity were designed. Results: Mutant (W38A) had reduced substrate affinity but a 2-fold increase in the maximum quasi-steady-state rate. Conclusion: Dissociation of stalled TrCel7A is the rate-limiting step in the initial phase of hydrolysis. Significance: This work offers a new perspective for the design of faster cellulases.

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“…GH7s contain a conserved tryptophan residue, Trp40 in TrCel7A, which binds to the glucopyranose ring at the −7 site in GH7 structures (31). This tunnel entrance tryptophan residue has been implicated in cellulose chain complexation (36,37), and its stacking interaction with the glucopyranose ring at the −7 site remains intact during the simulation. These results suggest that the Trp40 residue is responsible for acting as a large, hydrophobic platform for chain binding when the enzyme is bound to a cellulose crystal, and that this binding site delineates the decrystallized chain from the cellulose crystal.…”

Section: Simulations Predictmentioning

confidence: 99%

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Payne

Resch

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. biofuels | cellulase | post-translational modification | carbohydrate recognition

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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

Cited by 84 publications

References 36 publications

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

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

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

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