Characterization and substrate specificity study of the novel (R)-amine transaminase MagAT

Chiral amines, as essential chiral building blocks in drug synthesis, present a considerable challenge in biomanufacturing due to the requirement for highly efficient stereoselective synthesis. In this study, we successfully cloned and heterologously expressed a novel ( R )-amine transaminase, MagAT, from Mycolicibacterium agri . Systematic analysis showed optimal activity at pH 7.0, with the highest reaction rate occurring within 30 min at 50 ℃. However, considering overall thermal stability, 40℃ was selected as the operating temperature for subsequent experiments. Furthermore, the enzyme retained nearly 100% catalytic activity in the presence of 10% methanol, DMSO, and chloroform. Kinetic analysis demonstrated that MagAT possessed high substrate affinity, with Michaelis constants (K m ) of 5.13 ± 0.45 mM for ( R )-1-phenylethan-1-amine and 3.73 ± 0.34 mM for pyruvate. Substrate specificity studies indicated that MagAT efficiently catalyzed the conversion of C3-C8 aliphatic amines (e.g., ( R )-2-aminooctane) and monocyclic aromatic amines (e.g., ( R )-1-phenylethan-1-amine), whereas its catalytic activity significantly declined for sterically hindered substrates such as naphthylamine. 3D structural modeling with AlphaFold3 and molecular docking showed that steric hindrance from key residues in the O-pocket critically influences substrate specificity. This study not only provides an efficient biocatalyst for the biosynthesis of chiral amines but also establishes a theoretical basis for the rational design of transaminases with broader substrate adaptability.