Reprogramming of Phenylalanine Dehydrogenase Enables Synthesis of N -Alkyl-α-amino Acids

Chiral N -alkyl-α-amino acids are valuable building blocks in peptide modification and drug design, yet their asymmetric synthesis remains challenging. Here, we report a catalytic reprogramming strategy that transformed an ammonia-dependent phenylalanine dehydrogenase ( Qt PheDH) into a primary amine-utilizing biocatalyst for the asymmetric synthesis of N -alkyl-α-amino acids. Computational analysis reveals that mutations D127G and K92A reconstructed the ammonia-recognition network, redirecting the reductive amination pathway toward utilizing organic amines. The reshaped amine pocket accommodates various amines and exhibits a synergistic interaction with the substrate-recognition module. Moreover, in a cascade reaction starting from l -phenylalanine, the engineered Qt PheDH enabled efficient synthesis of diverse N -alkyl- l -phenylalanines, providing a strategy for reprogramming biocatalysts toward N -alkyl-α-amino acid synthesis.