A Mechanism-Guided Strategy by Weighting Key Reaction States for Rational Engineering of ω-Transaminase toward Bulky Chiral Amines

A mechanism-guided strategy weighting two key reaction states, the prereaction conformation and external aldimine, enabled rational engineering of a novel ω-transaminase ATA-116 for bulky chiral amines. Two residues, W57F and R390S, were identified, showing 210- and 107-fold improvements in k cat / K m, respectively (>99% ee). The highest conversion reached 91% (2.5% for the wild type). The strategy also expanded the substrate scope across 15 indanone derivatives and improved thermostability. Molecular dynamics simulations elucidated the mechanistic basis.