Semi-Rational Design of Substrate Channels for PaBVMO to Achieve Triple Synergy of Catalytic Efficiency-Substrate Inhibition-Stability

Baeyer-Villiger monooxygenase from Pseudomonas aeruginosa (PaBVMO) selectively inserts an oxygen atom into the unconventional side of the carbonyl group of 10-carbonyl stearic acid to produce octyl sebacate, which can be used for synthesizing material monomers sebacic acid and 8-hydroxyoctanoic acid. To break through the dual bottlenecks of low catalytic efficiency and significant substrate inhibition of PaBVMO under high substrate concentrations in sebacic acid biosynthesis, this study proposed a semi-rational remodeling strategy for the enzyme's substrate channel. By analyzing enzyme-substrate binding free energy and substrate force, the key gating loop structure of the substrate channel was resolved. Based on this strategy, the optimal mutant PaBVMO_G398A was obtained. It exhibited a catalytic turnover number (TON) of 3.45×10⁴, which was 5.3 times higher than that of the parent, and about 0.7 times higher in specific activity. Meanwhile, the inactivation half-life was prolonged from 1.87 h to 6.14 h, and the stability is significantly enhanced. Gaussian-accelerated molecular dynamics (GaMD) simulations further revealed the synergistic mechanism of G398A mutation inducing the rearrangement of the active pocket of PaBVMO_G398A. This rearrangement effectively alleviated substrate inhibition by 10-carbonyl stearic acid, enhanced affinity for FAD and improved stability.