Structural Analysis and Mutability Landscape-Guided Engineering of ω-Transaminase for Improved Catalytic Performance

ω-Transaminases (ω-TAs) are attractive biocatalysts in the asymmetric synthesis of chiral amines. Recently, a promising ω-TA from <i>Salmonella enterica</i> (<i>Se</i>TA) has been used to produce the valuable herbicide l-phosphinothricin (l-PPT). To improve the catalytic activity of <i>Se</i>TA, a combination strategy involving structural analysis and mutability landscape construction was conducted. A flexible region (loop1) enhancing the catalytic activity of <i>Se</i>TA was identified, and a distal mutant I22D (M1) was successfully screened. M1 showed a 3.24-fold increase in specific activity, along with greater thermal stability. Its half-life (<i>t</i>_1/2) was 30.95-fold (45 °C) and 17.53-fold (55 °C) longer than that of WT, and <i>T</i>_m increased by 7.6 °C. MD simulations revealed that the distal mutation induced long-range effects through allostery, resulting in the substrate tunnel being more spacious, reshaping the active pocket, and stabilizing the local conformation. These findings can provide insights into the distal sites and functional relationships of ω-TAs for improving their performance.