AlphaFold-Guided Semi-Rational Engineering of an (R)-Amine Transaminase for Green Synthesis of Chiral Amines

Chiral amines are vital structural motifs in pharmaceuticals and agrochemicals, where enantiomeric purity governs bioactivity and environmental behavior. We identified a novel ( R )-selective amine transaminase (MwoAT) from Mycobacterium sp. via genome mining, which exhibits activity toward the synthesis of the chiral amine ( R )-1-methyl-3-phenylpropylamine. The enzyme displayed optimal activity at pH 7.0 and 40 °C, with high thermostability and solvent tolerance. Using an AlphaFold3-guided semi-rational engineering strategy integrating molecular docking, alanine scanning, and saturation mutagenesis, residue L175 was pinpointed as critical for substrate binding. The resulting L175G variant exhibited a 2.1-fold increase in catalytic efficiency (k cat /K m ) and improved thermal stability. Applied to the asymmetric synthesis of ( R )-1-methyl-3-phenylpropylamine—a precursor for the antihypertensive drug dilevalol and potential scaffold for crop protection agents—the mutant achieved 26.4% conversion with ≥99.9% ee. The enzyme also accepted several ketones relevant to agrochemical synthesis, underscoring its versatility. This work delivers an engineered biocatalyst for sustainable chiral amine production and demonstrates an AI-assisted protein engineering framework applicable to both medicinal and agricultural chemistry.