Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose

Akshata R. Mudinoor,Jennifer Nill,Tina Jeoh,Alex Hitomi,Nardrapee Karuna,Raghavendra U. Rao,Peter M. Goodwin

doi:10.1186/s13068-020-1649-7

Abstract

BackgroundMolecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Qintact) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Qcore, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy.ResultsBinding of WTintact and E212Qintact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Qcore, exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized.ConclusionsCel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.

Highlights

Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters
The processive hydrolysis of cellulose by T. reesei Trichoderma reesei Cel7A (Cel7A) (Cel7A) entails multiple sequential steps: adsorption of the enzyme to the cellulose surface, surface diffusion, complexation wherein the catalytic domain recognizes and engages the reducing end of a single molecule of cellulose within its active site tunnel, glycosidic bond hydrolysis to form cellobiose as the product, product expulsion from the active site and subsequent sliding along the molecule to release several consecutive cellobioses, decomplexation and desorption from the cellulose surface [6]
We speculate that the carbohydratebinding module (CBM) and linker increases the affinity of Cel7A to cellulose possibly by facilitating recognition of complexation sites and by aiding in the uptake of a cellodextrin chain into the enzyme active site

Summary

Introduction

Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. Industrial cellulosic biofuel production processes employ fungal cellulase mixtures to breakdown cellulose into soluble sugars for further fermentation into fuels. Cellobiohydrolases (e.g., Cel7A of the well-characterized Trichoderma reesei) are the molecular workhorses that hydrolyze the recalcitrant cellulose in synergy with supporting endocellulase and oxidative activities [4, 5]. The different populations of bound Cel7A are challenging to distinguish in biochemical determinations of interaction rate parameters that are typically obtained from fitting hydrolysis or binding time courses, thereby resulting in broad ranges of values [14].

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