Mechanistic Insights into Enzyme Gate Engineering of ω-Transaminase for Regulating Organic Solvent Resistance

ω-Transaminases (ω-TAs) catalyze the synthesis of chiral amines in high-concentration organic solvents, enabling the sustainable production of agrochemicals, pharmaceuticals, and food additives. Previously, we developed an "enzyme gate engineering" strategy to enhance the organic solvent resistance of ω-TAs, although the underlying gating mechanism remained unclear. Herein, molecular dynamics simulations combined with multi-scale analyses of AcATA (an ω-TA from Arthrobacter cumminsii) revealed that substrate ketone interactions with switch residues Ile157 and Phe136 trigger hinge movements in loops 1-3, promoting contact between Gln155 and Phe56 to close the substrate-binding pocket entrance. We developed a Tunnel Dynamics Classification (TDC) method that analyzes how often tunnels of different sizes appear during MD simulations. To assess the gate effect of AcATA_M8 and its variants, we compared the tunnel frequencies between the substrate-free and substrate-binding states. The results showed a correlation of R = 0.70 with enzyme activity at 60% DMSO. Notably, the variant AcATA_M8^T134A exhibited only a 17% loss of enzymatic activity as the DMSO concentration increased from 20% to 60%, compared to a 35% reduction for AcATA_M8, and its half-life in 50% DMSO improved by 33%, successfully avoiding the typical "activity-stability trade-off".