Catalytic mechanism of a family 3 ß-glucosidase from the biomass-degrading fungus Hypocrea jecorina

Purpose and goal

The project aimed to comprehensively describe the catalytic reaction of H. jecorina GH3 Bgl. The specific objectives were to determine the kinetic properties of Bgl variants, elucidate the mechanisms of hydrolysis and the competing transglycosylation reaction, and interpret the pH dependence of each reaction. The study on the role of pH was recently published in Organic & Biomolecular Chemistry. Computational/experimental mechanistic studies have also been performed and are expected to be published later this year.

Expected results and effects

Quantum mechanics/molecular mechanics indicated the role of Asp61, Lys158, and Ser384 in stabilizing the transition state of the initial glycosylation step. Constant pH molecular dynamics showed that the pKa of the acid/base catalyst E441 is rather low due to hydrogen bonding with nearby arginine residues, which could reduce hydrolytic activity and favor transglycosylation. These findings would enable the protein engineering of more active variants that will improve efficiency of lignocellulolytic enzyme cocktails.

Planned approach and implementation

The proposed research plan was closely followed. Mechanisms were studied using quantum mechanics/molecular mechanics, while the pH-activity profile were modeled using constant pH molecular dynamics. Additional analyses were done using density functional theory. Calculations were primarily run on the Swedish National Infrastructure for Computing resources. Computational results were used to interpret experimental kinetic data obtained using high-performance anion exchange-chromatography with pulsed amperometric detection.