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 are 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. Database Structural data are available in RCSB Protein Data Bank database under the accession number https://www.rcsb.org/structure/6SU8. Enzymes ReCel7B, endoglucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from Rasamsonia emersonii; TrCel7B, endoglucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html) from Trichoderma reesei; TrCel7A, cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from Trichoderma reesei.

Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases

“…GH7s consist of two main subtypes, CBHs and EGs. Although over 5000 GH7 sequences are known, structural information is presently available for only 21 GH7s (16 CBHs, five EGs) ( 10 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ). GH7 CBH and EG structures share a similar β-jelly roll fold with two antiparallel β-sheets that pack into a curved β-sandwich ( 14 ).…”

Structural and functional characterization of a multi-domain GH92 α-1,2-mannosidase from Neobacillus novalis

“…Thus, the measured concentration of sugar reducing ends was assumed to be equal to the mannose reducing ends (actual yield). The yeast -mannan was acid hydrolysed, the released monosaccharides were quantified by HPAEC-PAD as described elsewhere (Schiano-di-Cola et al, 2020) and the mannose concentration was calculated. This concentration was divided by the initial yeast -mannan concentration corrected for the monomeric units (180/162) to obtain the theoretical yield.…”

“…This concentration was divided by the initial yeast -mannan concentration corrected for the monomeric units (180/162) to obtain the theoretical yield. The sample analyses using HPAEC-PAD followed the procedure described previously (Schiano-di-Cola et al, 2020).…”

Modulation of the catalytic activity and thermostability of a highly thermostable GH7 endoglucanase by engineering the key loop B3

“…By far ve crystal structures of GH7 endoglucanases have been resolved, including FoCel7B (Fusarium oxysporum; 1OVW), TrCel7B (Trichoderma reesei; 1EG1), HiCel7B (Humicola insolens; 6YOZ), ThCel7B (Trichoderma harzianum CBS 226.95; PDB ID: 5W0A),, and ReCel7B (Rasamsonia emersonii CBS 394.64; 6SU8). These structures share a common β jelly roll fold with two largely antiparallel β-sheets packing face to face to form a curved β-sandwich [16][17][18][19][20]. The catalytic cleft is covered by loops, in which the loop B3 (referred as the "exo" loop in the original publication) is located at the tunnel where the substrate binds, forming the roof of active site tunnel at the catalytic center [15].…”