Switching Enantioselectivity in Phenylalanine Ammonia Lyase for the Synthesis of Electron‐Deficient Aromatic <scp>d</scp>‐Amino Acids

Aromatic d‐amino acids (d‐AAs) are valuable building blocks in drug discovery and peptide therapeutics, yet their direct and efficient biocatalytic synthesis remains a challenge. Here, we report the rational engineering of phenylalanine ammonia lyase from Planctomyces brasiliensis (PbPAL) to enable asymmetric hydroamination for the enantioselective synthesis of d‐aromatic amino acids. By targeting active‐site residue L205, we identified variants capable of highly d‐enantioselective hydroamination, with L205F enabling the transformation of electron‐deficient aryl acrylates with >99% enantiomeric excess (ee). The synthetic utility of this platform was demonstrated by gram‐scale synthesis of d‐benzoxazole and substituted 2‐pyridylalanines. Structural and mutational studies revealed distinct roles for the 4‐methylideneimidazole‐5‐one (MIO) prosthetic group and active‐site residues L205, Y64, and K397 in modulating enantioselectivity. These results enabled the identification of PbPAL variants with the opposite selectivity, such as L205V‐K397A, which preferentially produce l‐amino acids. This work broadens the utility of PALs as programmable biocatalysts for asymmetric synthesis.