Mapping the Hydrophobic Substrate Binding Site of Phenylalanine Ammonia-Lyase from Petroselinum crispum

Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity toward phenylalanines and cinnamic acids monosubstituted with both electron-donating (−CH3, −OCH3) and electron-withdrawing (−CF3, −Br) groups at all positions (ortho, meta, para) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at the ortho, meta, and para positions of the substrate's phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of p-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative-scale synthesis of (S)-m-bromo-phenylalanine (ee > 99%; yield 60%) and (R)-p-methyl-phenylalanine (ee 97%; yield 49%) using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure-based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.