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 substratebinding 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.

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…As already stated, the high/low values of substrate loads are well above/below K M for both Avicel and pNPL, and the data therefore reasonably represent the limiting cases. This interpretation is further supported by published binding parameters for the same enzyme–substrate systems (Badino, Kari, Christensen, Borch, & Westh, ; Schiano‐di‐Cola et al, ; Westh et al, ), which suggests almost complete adsorption of enzyme (85–98% bound) at high Avicel load (90 g/L). We used nonlinear regression analyses to fit the pH curves as detailed in the Supporting information.…”

Systematic modification of N-glycosylation sites in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

“…3 C, Table 3) shows that the N45 Nglycans not only hinder the ability to form productive complexes on the cellulose surface, it also decreases the overall adsorption. The negative impact of the N45 N-glycans on Γ max and Γ attack could be further explained by the fact that TrCel7A loops, shaping the catalytic tunnel, play a key role in modulating the enzyme's ability to attack cellulose surface [30] and kinetics [31]. Thus, a bulky, heterogeneous glycan structure on the entrance loop might lead to reduced productive binding on the cellulose surface.…”

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

“…Reactions were terminated by a 3 min centrifugation at 2000 g . The concentration of soluble reducing ends was then determined from the supernatant using the para ‐hydroxybenzoic acid hydrazide (PAHBAH) method , with a procedure described elsewhere . The concentration of the soluble sugar reducing ends was quantified against a 6‐point cellobiose calibration curve in a concentration range between 31.25 and 1000 µ m .…”

“…The reaction was stopped by a 3‐min centrifugation at 2000 g . Volumes of 60 μL of the supernatant were retrieved and analyzed for its content of reducing sugars by using the 4‐hydroxybenzoic acid hydrazide (PAHBAH) method as described before . Finally, 100 μL was transferred into a microtiter plate, and the absorbance at 405 nm was measured by a plate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA).…”

“…The EGs have fewer subsites and a more exposed catalytic cleft, as a consequence of the shorter loops covering this region . It has been shown earlier that the length of the loops covering the catalytic cleft plays a significant role in tuning processivity, binding, and endolytic activity of GH7 enzymes .…”