The Structure of a Bacterial Cellobiohydrolase: The Catalytic Core of the Thermobifida fusca Family GH6 Cellobiohydrolase Cel6B

Sandgren, Mats; Wu, Miao; Karkehabadi, Saeid; Mitchinson, Colin; Kelemen, Bradley R.; Larenas, E.; Ståhlberg, Jerry; Hansson, Henrik

doi:10.1016/j.jmb.2012.11.039

Cited by 38 publications

(36 citation statements)

References 52 publications

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“…In this study, we extend our previous studies on TfuCel6B (23,29). TfuCel6B is an interesting GH6 model system because of its extended and more enclosed tunnel, and also because Wilson and co-workers (33,34) have conducted considerable experimental work on its catalytic mechanism, substrate specificity, and synergy with other T. fusca enzymes, thus providing significant mutational data for mechanistic interpretations.…”

supporting

confidence: 59%

“…To remove the carbohydrate-binding module, the full-length enzymes were subjected to papain digestion, and the resulting catalytic domains were purified using ion exchange chromatography on a Source 30Q column as described previously (23) followed by size-exclusion chromatography on a Superdex 200 column.…”

Section: Methodsmentioning

confidence: 99%

“…Crystallization and Data Collection-We co-crystallized the catalytic domains of TfuCel6B variants (6 mg/ml) with 10 mM cellobiose using the hanging drop method as described previously (23). The gradient seeding method was necessary for obtaining crystals and improving quality of the crystals.…”

Section: Methodsmentioning

confidence: 99%

“…Fig. 1 shows a surface representation of known structures of GH6 CBHs (22)(23)(24)(25) and endoglucanases (26,27). The T. fusca GH6 enzymes represent the extremes of known structural diversity in terms of active site enclosure.…”

mentioning

confidence: 99%

“…Instead, the predominant mechanism for GH6 hydrolysis proposed to date from several structural studies involves a water wire or Grotthuss mechanism. Several structures reveal a nucleophilic water molecule near the anomeric carbon stabilized by a serine residue on the "active center" loop (residues 175-183 in HjeCel6A and 226 -234 in TfuCel6B) (23,30). This water molecule is hydrogen-bonded to a second water molecule, which in turn hydrogen-bonds to a conserved aspartate (Asp-175 in HjeCel6A and Asp-226 in TfuCel6B).…”

mentioning

confidence: 99%

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Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Vuong

et al. 2013

Journal of Biological Chemistry

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Background: Family 6 glycoside hydrolases represent an important, diverse enzyme class in cellulolytic organisms. Results: We solved structures of two Thermobifida fusca Cel6B (TfuCel6B) cellobiohydrolase mutants and examined ligand dynamics and product release with simulation. Conclusion: These results suggest mechanisms for product release in TfuCel6B. Significance: This study further elucidates the mechanism of a unique cellobiohydrolase with an extended, enclosed active site tunnel.

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supporting

confidence: 59%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Vuong

et al. 2013

Journal of Biological Chemistry

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Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases

Hefferon

Cantero-Tubilla

Brady

et al. 2019

Biotech & Bioengineering

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Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes. K E Y W O R D Sactive site tunnel, exocellulase, recalcitrant cellulose, synergism, Thermobifida fusca

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CAZyme Characterization and Engineering for Biofuels Applications

Nemmaru,

DeChellis,

Patil

et al. 2024

Handbook of Biorefinery Research and Technology: Biomass Logistics to Saccharification

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The Structure of a Bacterial Cellobiohydrolase: The Catalytic Core of the Thermobifida fusca Family GH6 Cellobiohydrolase Cel6B

Cited by 38 publications

References 52 publications

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases

CAZyme Characterization and Engineering for Biofuels Applications

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