Substrate-pocket reshaping of an (R)-selective transaminase enables efficient asymmetric synthesis of (R)-3-amino-1-butanol.

(R)-Selective ω-transaminases are valuable biocatalysts for the synthesis of chiral amines and pharmaceutical intermediates, but their practical application is often limited by insufficient catalytic efficiency, poor operational stability and unfavorable reaction equilibrium. Here, an Actinomycete-derived (R)-selective ω-transaminase, AcTA, was engineered for the asymmetric reductive amination of 4-hydroxy-2-butanone to produce (R)-3-amino-1-butanol. Through a combination of molecular modeling, alanine scanning, consensus analysis and iterative saturation mutagenesis, the substrate-binding pocket of AcTA was reshaped to improve substrate positioning and catalytic performance. The optimal triple variant M3 (H73R/A168V/S210R) exhibited a 13.74-fold increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. M3 also showed markedly enhanced thermostability, with T₅₀10, t₁/₂40 and Tm values increased by 15.22 °C, 7.97 min and 6.46 °C, respectively. Under optimized reaction conditions, M3 converted 50 g/L 4-hydroxy-2-butanone with 94.28% conversion and 99.5% ee within 36 h. These results establish M3 as an efficient and thermostable biocatalyst for the asymmetric synthesis of (R)-3-amino-1-butanol and demonstrate that substrate-pocket reshaping is an effective strategy for improving (R)-selective transaminases.