Methyltransferase “Gating Loop” Reengineering: Reshaping Catalytic Performance through Tunnel Dynamics and Structural Flexibility

can catalyze the N-terminal trimethylation of L-tryptophan to produce the medicinal indole alkaloid L-hypaphorine. Structural comparison, loop deletion, and alanine scanning revealed that Loop34-39 and Loop163-168 are critical for MsE activity and substrate binding. Saturation and iterative saturation mutagenesis targeting hotspot residues in these loops yielded mutant M5 (P35C/K36R/F38L/T163G/T168F). Compared with the original enzyme MsE, M5 exhibits a 7.11-fold increase in relative activity and achieves a conversion rate of 58.93%. Molecular dynamics simulations, CAVER tunnel analysis, and ligand dissociation pathway studies reveal that M5 modulates the hydrogen bond network of the active site and remodels the conformation of the substrate access tunnel, thereby transforming the substrate tunnel into a methylation reaction microcompartment with a "capture-release" function. This study provides a theoretical and practical basis for loop optimization engineering and enzyme functional modification.