Crystal Structure of a GH3 β-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum

Abstract

Beta-glucosidases (β-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 β-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other β-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family β-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in β-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

Keywords: Chaetomium thermophilum; cellulose degradation; fungal enzymes; glycoside hydrolase; protein structure; thermophilic fungus; β-glucosidases.

Figure 1

Structure-based sequence alignment of CtBGL with members of GH3 family. Secondary structure elements are shown on the top of the alignment, while the red triangles below the alignment indicate the catalytic conserved residues (Asp287 and Glu517 in CtBGL). Disulfide bonds are depicted with green numbers. The three domains are indicated. A column is framed if more than 70% of its residues are similar according to physicochemical properties. Frames in red background with white letters depict strict identity. The figure was constructed with ESPript [16].

Figure 2

Overall crystal structure of CtBGL in ribbon representation. The domains, linkers, and loops are shown in different colors and labeled: Catalytic TIM barrel-like domain (blue), α/β sandwich domain (green), FnIII domain (yellow), loop I (gold), linker 1 (cyan), loop II or insertion region (magenta), loop III (orange), loop IV (orange–red), linker 2 (gray), loop V (brown). The N-glycans and glucose in the active site are shown as gray sticks. The figure was created with UCSF Chimera [17].

Figure 3

Overview of domain 2 in ribbon representation CtBGL superimposed with the compared structures. Domain 2 is colored orange; loops III and IV in CtBGL, NcCel3A, ReCel3A, and TnBgl3B are colored in green, blue, cyan, and yellow, respectively, for structural comparisons. Linker 2 loop is colored in black, and the active site is depicted by the bound BGC in sphere representation.

Figure 4

Loop V comparisons in (a) CtBGL, (b) NcCel3A, (c) ReCel3A, (d) AaBgl1, (e) HjCel3A, and (f) TnBgl3B. Domain 3 is colored in yellow and the loop V is represented by red coloration. Domains 1 and 2 are colored in the same color in each enzyme and differently amongst the enzymes.

Figure 5

Distribution of glycosylation sites in the CtBGL structure. GlcNAc is shown in blue, β-d-mannose in green, α-d-mannose in orange, and BGC in magenta.

Figure 6

Active site structure comparisons of (a) CtBGL, (b) NcCel3A, (c) ReCel3A, (d) TnBgl3B, (e) HjCel3A, and (f) AaBGL1. The corresponding conserved nucleophile Asp is colored in dark green and the acid/base Glu is colored in yellow. Both residues are labeled in red. Carbon atoms are colored differently in each enzyme. The active site BGC is displayed with its solvent-excluded surface for visual clarity.