Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

Corinna Schiano di Cola, Bartlomiej Kolaczkowski, Trine Holst Sørensen, Stefan Jarl Christensen, Ana Mafalda Cavaleiro, Michael Skovbo Windahl, Kim Borch, Jens Preben Morth, Peter Westh

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data is available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate binding module (CBM). With a temperature optimum of 80°C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate binding cleft. An increased number of glycosylations were identified in ReCel7B and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding and cellulose accessibility in GH7 endoglucanases.
OriginalsprogEngelsk
TidsskriftF E B S Journal
ISSN1742-464X
DOI
StatusUdgivet - 21 nov. 2019

Citer dette

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title = "Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii",
abstract = "Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data is available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate binding module (CBM). With a temperature optimum of 80°C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 {\AA} resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate binding cleft. An increased number of glycosylations were identified in ReCel7B and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding and cellulose accessibility in GH7 endoglucanases.",
author = "Cola, {Corinna Schiano di} and Bartlomiej Kolaczkowski and S{\o}rensen, {Trine Holst} and Christensen, {Stefan Jarl} and Cavaleiro, {Ana Mafalda} and Windahl, {Michael Skovbo} and Kim Borch and Morth, {Jens Preben} and Peter Westh",
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language = "English",
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Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii. / Cola, Corinna Schiano di; Kolaczkowski, Bartlomiej; Sørensen, Trine Holst; Christensen, Stefan Jarl; Cavaleiro, Ana Mafalda; Windahl, Michael Skovbo; Borch, Kim; Morth, Jens Preben; Westh, Peter.

I: F E B S Journal, 21.11.2019.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

AU - Cola, Corinna Schiano di

AU - Kolaczkowski, Bartlomiej

AU - Sørensen, Trine Holst

AU - Christensen, Stefan Jarl

AU - Cavaleiro, Ana Mafalda

AU - Windahl, Michael Skovbo

AU - Borch, Kim

AU - Morth, Jens Preben

AU - Westh, Peter

PY - 2019/11/21

Y1 - 2019/11/21

N2 - Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data is available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate binding module (CBM). With a temperature optimum of 80°C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate binding cleft. An increased number of glycosylations were identified in ReCel7B and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding and cellulose accessibility in GH7 endoglucanases.

AB - Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data is available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate binding module (CBM). With a temperature optimum of 80°C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate binding cleft. An increased number of glycosylations were identified in ReCel7B and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding and cellulose accessibility in GH7 endoglucanases.

U2 - 10.1111/febs.15151

DO - 10.1111/febs.15151

M3 - Journal article

JO - F E B S Journal

JF - F E B S Journal

SN - 1742-464X

ER -