Structure-Guided\nEngineering Unveils Deeper Substrate\nChannel in Processive Endoglucanase EG5C‑1 Contributing to\nEnhanced Catalytic Efficiency and Processivity

Processive endoglucanases have generated significant\ninterest due\nto their bifunctionality in the degradation of cellulose and low product\ninhibition. However, enhancing their catalytic efficiency through\nengineering remains a formidable challenge. To address this bottleneck,\nour engineering efforts targeted loop regions located in the substrate\nchannel of processive endoglucanase EG5C-1. Guided by a comparative\nanalysis of characteristic structural features of the substrate channels\nin cellobiohydrolase, endoglucanase, and processive endoglucanase,\na highly active triple mutant CM6 (N105H/T205S/D233L) was generated\nthat had a 5.1- and 4.7-fold increase in catalytic efficiency toward\nsoluble substrate carboxymethyl cellulose-Na and insoluble substrate\nphosphoric acid-swollen cellulose (PASC), compared with wild-type\nEG5C-1. Furthermore, this mutant exhibited greater processivity compared\nto EG5C-1. Molecular dynamics simulations unveiled that the mutations\nin the loop regions reshaped the substrate channel, leading to a deeper\ncleft, resembling the closed channel configuration of cellobiohydrolases.\nThe increased compactness of the substrate channel induced changes\nin the substrate binding mode and substrate deformation, thereby enhancing\nboth binding affinity and catalytic efficiency. Moreover, metadynamics\nsimulations demonstrated that the processive velocity of cellulose\nchain through the binding channel in mutant CM6 surpassed that observed\nin EG5C-1.