Arg128*‐Mediated Dual‐Substrate Recognition and Dynamic Transport Mechanisms in (R)‐ω‐Transaminase: Computational Insights and Mutational Profiling Guided Rational Engineering

ω-Transaminases (ω-TAs) are critical biocatalysts for the asymmetric synthesis of chiral amines, and uniquely accommodate both hydrophobic and hydrophilic substrates through a conserved binding pocket. In this study, we combine computational simulations and site-directed mutagenesis to dissect this dual-function structure of (R)-selective ω-transaminase from Aspergillus terreus (AtATA). Our results reveal that AtATA employs a synergistic mechanism: aromatic residues within the large pocket stabilize hydrophobic substrates via π-driven interactions, while Arg128* dynamically interacts with hydrophilic compounds through hydrogen bonding. Furthermore, the binding pocket of AtATA exhibits remarkable plasticity to accommodate diverse substrates, with the side chain of Arg128* dynamically adjusting its conformation to facilitate the transport of substrates. Mutational profiling, particularly the R128*A mutation, directly validates these mechanistic insights. Our finding reveals the Arg128*-mediated dual-substrate recognition and transport mechanisms, providing a solid theoretical foundation for enhancing the industrial application of transaminases in pharmaceutical synthesis and green chemistry.