QM/MM investigation of the catalytic mechanism of processive endoglucanase Cel9G from .

Carbohydrate degradation catalyzed by glucoside hydrolases (GHs) is a major mechanism in biomass conversion. GH family 9 endoglucanase (Cel9G) from , a typical multimodular enzyme, contains a catalytic domain closely linked to a family 3c carbohydrate-binding module (CBM3c). Unlike the conventional behavior proposed for other carbohydrate-binding modules, CBM3c has a direct impact on catalytic activity. In this work, extensive molecular dynamics (MD) simulations were employed to clarify the functional role of CBM3c. Furthermore, the detailed catalytic mechanism of Cel9G was investigated at the atomistic level using the combined quantum mechanical and molecular mechanical (QM/MM) method. Based on these simulations, owing to the rigidity of the peptide linker, CBM3c may affect the enzymatic activity direct interactions with alpha helix 4 of GH9, especially with the K123 and H125 residues. In addition, using cellohexaose as a substrate, the QM/MM MD simulations confirmed that this enzyme can cleave the β-1,4-glycosidic linkage an inverting mechanism. An oxocarbenium ion-like transition state was located with a barrier height of 19.6 kcal mol. Furthermore, the G(-1) pyranose unit preferentially adopted a distorted S/H conformer in the enzyme-substrate complex. For the cleavage of the glycosidic bond, we were able to identify a plausible route (S/H → [H/E] → C) from the reactant to the product at the G(-1) site.