Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part II. Structural Analysis of Thermotolerant Mutant from the Basidiomycete Phanerochaete chrysosporium

Abstract

Glycoside hydrolase family 6 cellobiohydrolase (GH6 CBH) is a group of cellulases capable of hydrolyzing crystalline cellulose. However, the synergistic reaction of GH6 CBH with other cellulases is hindered by its relatively low thermotolerance. We previously obtained a thermotolerant double mutant, C240S/C393S, of GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCel6A) by replacing the two free cysteine (Cys) residues, C240 and C393, with serine (Yamaguchi et al., J Appl Glycosci. 2020; 67;79-86). In the accompanying paper (Part I; Yamaguchi et al., J Appl Glycosci. 2024; 71: 55-62), we measured the temperature dependence of the activity and folding of C240S/C393S and its single mutants, C240S and C393S, and found that replacement of C393 was the major contributor to the increased thermotolerance of C240S/C393S. Here, in order to investigate the mechanism involved, we crystallized the wild-type and the mutant enzymes and compared their X-ray crystal structures. The overall structures of the wild-type and the three mutant enzymes were similar. However, C240S/C393S had the lowest relative B-factor at both the N-terminal loop (residues 172-177) and the C-terminal loop (residues 390-425). This result suggests that reduced structural fluctuation of the substrate-enclosing loops, possibly due to stronger hydrogen bonding involving C393, could account for the increased thermotolerance of C240S/C393S.

Keywords: Cellulose; GH6; X-ray crystal structure; cellobiohydrolase; free cysteine; thermotolerance.

Fig. 1.. B-factors of each residue's main chain (A) and side chain (B) normalized to the chain mean (WT, blue; C240S, green; C393S, yellow; C240S/C393S, red).

Arrowheads represent the positions of the 240th and 393rd amino acids.

Fig. 2.. B-factors in the crystal structures of PcCel6A (A) WT, (B) C240S, (C) C393S, and (D) C240S/C393S.

The warmer colors and bolder lines represent larger B-factors normalized in each structure. The figures below represent the views of the upper figures from the right side.

Fig. 3.. Ensemble models of the crystal structures of PcCel6A WT (blue), C240S (green), C393S (yellow), and C240S/C393S (pink). The N- and C-terminal loops are shown in darker color.

(A) The side chains of Cys residues forming disulfide bonds, the 240th and 393rd amino acids, and Q187 are shown as sticks in the overall structure. (B) Residues within 4 Å from the 240th amino acid. (C) Extracted view of 240th amino acid and the adjacent Q187. Distance (Å) between the sidechains in a static structure is indicated by a dotted line and a single decimal number. (D) Residues within 4 Å from the 393rd amino acid and the C361-C408 disulfide bond. (E) Extracted view of a pathway from the 393rd amino acid to the C361-C408 disulfide bond via N362. Distance (Å) between the sidechains in a static structure is indicated by dotted lines and single decimal numbers.

Fig. 4.. Histograms of the dihedral angles of the 240th and 393rd amino acids and some neighboring residues in the ensemble models of WT (A), C240S (B), C393S (C), and C240S/C393S (D).

Fig. 5.. Carboxylic acid pairs in PcCel6A.

(A) Potential carboxyl-carboxylate hydrogen bond sites. Ensemble models of the pairs and free Cys residues are shown as sticks. The N- and C-terminal loops are colored darker. The bold one-letter alphabet indicates the name of the enlarged figure seen from the arrowhead. (B-E) Magnified view of each carboxylic acid pair in WT (blue), C240S (green), C393S (yellow), and C240S/C393S (pink).

Fig. 6.. Interaction of the C393 side chain with the main chain of neighboring residues.

(A) The close-up view around C393 in WT. The dashed line is the measured length from the side chain of the 393rd amino acid to the main-chain nitrogen of D394 (I) and G395 (II), and the main-chain oxygen of G395 (III) and P418 (IV). (B) Distance between the side chain of the 393rd amino acid of WT (blue), C240S (green), C393S (yellow), and C240S/C393S (red) with the main chains of neighboring residues illustrated in the structure.

Fig. 7.. Conservation of free Cys among characterized GH6.

The colors of the node tips indicate fungal GH6 described as EC 3.2.1.91 in CAZy (blue), eukaryotic GH6 for which EC 3.2.1.4 activity was reported (green), and bacterial GH6 described as EC 3.2.1.91 in CAZy (orange), GH6 from an anaerobic fungus characterized as EC 3.2.1.91 in CAZy (light blue), GH6 from an anaerobic fungus for which EC 3.2.1.4 was reported (light green), bacterial GH6 described as EC 3.2.1.4 in CAZy (red), GH6 from an unclassified organism (gray), and GH6 indicated as other than EC 3.2.1.4 and 3.2.1.91 in CAZy (black). The first and second letters before the file name represent the amino acids at the positions corresponding to the 240th and 393rd free Cys residues in PcCel6A. The label of GH6 CBH from an aerobic thermophilic fungus is written in dark orange. The abbreviated names of some well-characterized GH6s are shown after the arrowheads: TrCel6A (Trichoderma reesei CBH), HiCel6A Humicola insolens CBH), CcCel6A (Coprinopsis cinerea CBH), HiCel6A (H. insolens EG), CcCel6C (one of C. cinerea EG), CfCel6B (Cellulomonas fimi CBH), TfCel6B (Thermobifida fusca CBH), OrpCelF (one of the Orpinomyces sp. CBHs), TfCel6A (T. fusca EG), and MtCel6 (Mycobacterium tuberculosis EG).