Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A

Abstract

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.

Keywords: Trichoderma reesei; binding; carbohydrate-binding protein; cellobiohydrolase; cellulase; cellulose; off-rate; on-rate; processivity; synergism.

FIGURE 1.

Structure and mechanism of TrCel7A. A, schematic representation (light blue) of catalytic domain, linker, and CBM for TrCel7A in complex with a cellulose strand. The O-glycosylation of the linker is shown as a surface representation and the cellulose strand is represented as green sticks. The image was made using the crystal structure of the catalytic domain (Protein Data Bank code 8CEL) and CBM (Protein Data Bank code 1CBH). B, positions of the Trp residues in the active site tunnel of TrCel7A in complex with a cellodextrin chain (green). Numbers refer to the different binding subsites in the tunnel with −7 at the entrance and −1/+1 being the position of the scissile bond. Trp-38 investigated in this study is highlighted in magenta. C, molecular steps in the hydrolysis of cellulose for a processive enzyme. Steps leading from the free enzyme in the solution to the enzyme with the reducing end of the cellulose chain in the −1 binding site are collectively referred to as feeding. Processive catalysis includes the formation of a Michaelis complex (by sliding the chain end from binding site −1 to +2), hydrolysis of glycosidic bond, and expulsion of cellobiose (green ellipses). Processive catalysis is repeated until the enzyme meets an obstacle (depicted here as upper cellulose fibril) or happens to dissociate.

FIGURE 2.

SEE with Avicel. In SEE, Avicel (100 mg ml⁻¹) was preincubated with 400 nm TrCel7A for 1 h, after which an equal volume of [¹⁴C]AC (final concentration 2 mg ml⁻¹) was added, and the release of radioactivity (expressed in [¹⁴C]CB equivalents) in time was followed (♢). In reference time curves (♦) the same conditions were used as in SEE but Avicel was mixed with [¹⁴C]AC before the addition of TrCel7A. Control experiments (□) of hydrolysis of [¹⁴C]AC (2 mg ml⁻¹) in the absence of Avicel are also shown (made in one parallel). Error bars show S.D. and are from two independent experiments. Solid lines represent the best fit from non-linear regression analysis (see “Experimental Procedures”). The TrCel7A variants used were: A, WT; B, W38A; C, WT_CD; D, W38A_CD.

FIGURE 3.

Formation of soluble and insoluble reducing groups in hydrolysis of rBC. rBC (1 mg ml⁻¹) was incubated with 100 nm TrCel7A and the formation of SRGs (A) and IRGs (B) in time was followed. Solid lines in B for the series with WT and WT_CD are from linear regression and the slopes were used to calculate the values of k_IRG. In panel C, the data are plotted in coordinates [RG]_tot versus [IRG]. Solid lines are from linear regression and the slope equals the P_app value. Error bars show S.D. and are from three independent experiments. With W38A, experiments were also made using 62 nm enzyme concentrations (▴).

FIGURE 4.

Formation of soluble and insoluble reducing groups in hydrolysis of rAC. rAC (1 mg ml⁻¹) was incubated with 100 nm TrCel7A and the formation of SRGs (A) and IRGs (B) in time was followed. Solid lines in B are from liner regression and the slopes were used to calculate the values of k_IRG. In panel C, the data are plotted in coordinates [RG]_tot versus [IRG]. Solid lines are from linear regression and the slope equals the P_app value. Error bars show S.D. and are from three independent experiments.

FIGURE 5.

Binding of WT TrCel7A and its W38A variant to BC. Total bound, WT (■) and W38A (♦); active site bound WT (□) and W38A (♢); bound enzyme with free active site, WT (▵) and W38A (▴). The concentration of total bound enzyme ([TrCel7A]_bound-tot) was found as a difference between the total enzyme concentration and the concentration of enzyme free in the solution. Concentration of active site bound enzyme ([TrCel7A]_bound-OA) was found from the strength of inhibition of MUL hydrolysis by BC. Concentration of bound enzyme with the free active site ([Cel7A]_bound-FA) was found as a difference between [TrCel7A]_bound-tot and [TrCel7A]_bound-OA. Error bars show S.D. and are from three independent experiments.

FIGURE 6.

The active site mediated binding of TrCel7A and its variants to AC. A, binding was measured with 5 nm total concentration of TrCel7A ([E]_tot) by varying the concentration of AC between 0.001 and 5 g liter⁻¹. Inset shows the enlargement of the region of low AC concentrations. Solid lines represent the best fit of non-linear regression according to Equation 2. B, binding data from panel A re-plotted in coordinates of concentration of the active site bound enzyme versus the concentration of enzyme with free active site. Solid lines represent the best fit of non-linear regression according to one binding site Langmuir isotherm. Error bars show S.D. and are from three independent experiments.

FIGURE 7.

Synergistic hydrolysis of BC by TrCel7A or its W38A variant in the presence of EG TrCel5A. A, release of radioactivity (in [¹⁴C]CB equivalents) in hydrolysis of [¹⁴C]BC by 250 nm TrCel7A acting in isolation, WT (■) and W38A (♦), or in the presence of 25 nm EG, WT + EG (□), and W38A + EG (♢). The release of radioactivity by 25 nm EG is also shown (x). B, the hydrolysis of native (filled labels) and EG pre-treated (open labels) BC by 100 nm TrCel7A. TrCel7A variants were: WT (■ and □), and W38A (♦ and ♢). The rate of cellulose hydrolysis (v_CB) was found from the formation of soluble reducing groups (in CB equivalents) after 1 h of hydrolysis. Error bars show S.D. and are from three independent experiments.