TY - JOUR
T1 - Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii
AU - Schiano di Cola, Corinna
AU - Kołaczkowski, Bartłomiej
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 - 2020
Y1 - 2020
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.
KW - Thermostable cellulase
KW - Endoglucanas
KW - Enzyme kinetics
KW - Cellulose
KW - Synergy
U2 - 10.1111/febs.15151
DO - 10.1111/febs.15151
M3 - Journal article
C2 - 31755197
SN - 1742-464X
VL - 287
SP - 2577
EP - 2596
JO - FEBS Journal
JF - FEBS Journal
IS - 12
ER -