Systematic deletions in the cellobiohydrolase (CBH) Cel7A from the fungus Trichoderma reesei reveal flexible loops critical for CBH activity

Abstract

Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure-function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites -3 and -4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.

Keywords: Cel7A; Cel7B; Trichoderma reesei; cellobiohydrolase; cellulase; cellulose; endoglucanase; enzyme kinetics; loop engineering; protein engineering.

Figure 1.

Structure of TrCel7A and TrCel7B with highlighted loops and sequence alignments. A, cartoon representation of TrCel7A (PDB entry 4C4C) and TrCel7B (PDB entry 1EG1) with cellononaose in yellow sticks. The substrate structure was taken from 4C4C and superimposed onto TrCel7B. The loops that cover the substrate-binding tunnel or cleft are highlighted in different colors. Loops that are common between the two enzymes are highlighted in black (A1–A3 and B1 loops). Loops unique to TrCel7A are highlighted in different colors: A4 loop (green), B2 loop (cyan), B3 loop (orange), and B4 loop (red). B, amino acid sequence alignments of the different loop regions of TrCel7A, loop deletion (Δ) variants of TrCel7A, and TrCel7B. The deletion mutants lack the residues Asn³⁸⁴–Ser³⁸⁸ for ΔA4, Trp¹⁹²–Gly²⁰⁵ for ΔB2-1, Glu¹⁹³–Gly²⁰⁵ for ΔB2-2, Ser¹⁹⁶–Thr²⁰¹ for ΔB2-3, Gly²⁴⁵–Gly²⁵³ for ΔB3, and Glu³³⁵–Phe³³⁸ for ΔB4. The dashed lines indicate the regions missing in the different sequences compared with TrCel7A. The loop nomenclature and the color coding are the same as in A. The loops of interest are illustrated on the right.

Figure 2.

Conventional Michaelis–Menten, inverse Michaelis–Menten plots, and binding isotherms for TrCel7A (red), TrCel7B (brown), and deletion variants ΔB2-1 (green), ΔB2-2 (purple), ΔB2-3 (gray), ΔB3 (pink), and ΔB4 (light blue). Steady-state initial rates of hydrolysis are plotted against the Avicel substrate load (S₀) at a constant enzyme concentration (A) (conditions: 0.5–110 g/liter Avicel, 50 nm enzyme, pH 5, 60 min, 25 °C) or against the enzyme concentration (E₀) at a constant substrate load (B) (conditions: 8 g/liter Avicel, 0.05–6 μm enzyme, pH 5, 60 min, 25 °C). Binding isotherms on Avicel (C) are performed in the same conditions as B. Symbols, experimental data with S.D. (error bars); lines, best fit for the different equations.

Figure 3.

Specific activity (v/E₀) on cellulosic substrates with different degree of crystallinity. Conditions were as follows: 4 g/liter RAC, 50 nm enzyme or 4 g/liter BMCC, 100 nm enzyme, 25 °C, 60 min. Error bars, S.D.