Structure-Guided Engineering Unveils Deeper Substrate Channel in Processive Endoglucanase EG5C-1 Contributing to Enhanced Catalytic Efficiency and Processivity

Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.