Ring-Substituted α-Arylalanines for Probing Substituent Effects on the Isomerization Reaction Catalyzed by an Aminomutase

Abstract

β-Amino acids are emerging as an important class of compounds in medicinal chemistry. β-Aryl-β-alanines show antiepileptogenesis activity, while others have been used to synthesize antibiotic β-peptides. To assess the utility of a methylidene imidazolone-dependent Pantoea agglomerans phenylalanine aminomutase (PaPAM) for making non-natural β-amino acids, we surveyed the substrate specificity of PaPAM with several commercially available (S)-arylalanine substrates. Here, we report the Michaelis–Menten parameters and catalytic efficiency of PaPAM for each substrate. Compared to phenylalanine, substrates containing substituents that were either electron-withdrawing or -donating through resonance or inductive effects affected the kcat of PaPAM. Generally, the turnover and catalytic efficiency of PaPAM for the meta-isomers were better than for the corresponding para- and ortho-isomers, with some exceptions. PaPAM principally synthesizes the β-amino acids at >90% and the cinnamate byproducts at <10% for 11 of the 19 productive substrates. The yield from other substrates was 14–65% of the cinnamate analogue. Further, to explain the determinants of substrate selectivity of PaPAM, a series of substrates with substituents on the aryl ring were docked into the crystal structure of the active site. Induced fit of the protein to accommodate different substituents was modeled computationally by SLIDE docking and Szybki energy minimization. The results provide insights into the roles of substrate orientation and conformational flexibility in turnover and indicate which terms of the interaction energy account for the experimentally observed KM values, which largely determine catalytic efficiency. Substrate selectivity of PaPAM is significantly influenced by steric barriers created by specific active-site residue interactions with the substituted aryl portion of the substrate.