Ancestral l-amino acid oxidase: From substrate scope exploration to phenylalanine ammonia-lyase assay

In this study we assessed the applicability of the recently reported ancestral l-amino acid oxidase (AncLAAO), for the development of an enzyme-coupled phenylalanine ammonia-lyase (PAL) activity assay. Firstly, the expression and isolation of the AncLAAO-N1 was optimized, followed by activity tests of the obtained octameric N-terminal His-tagged enzyme towards various phenylalanine analogues to assess the compatibility of its substrate scope with that of the well-characterized PALs. AncLAAO-N1 showed high catalytic efficiency towards phenylalanines mono-, di-, or multiple-substituted in the meta- or para-positions, with ortho- substituted substrates being poorly transformed, these results highlighting the significant overlap between its substrate scope and those of PALs. After successful set-up of the AncLAAO-PAL coupled solid phase assay, in a 'proof of concept' approach we demonstrated its applicability for the high-throughput activity screens of PAL-libraries, by screening the saturation mutagenesis-derived I460NNK variant library of PAL from Petroselinum crispum, using p-MeO-phenylalanine as model substrate. Notably, the hits revealed by the coupled assay comprised all the active PAL variants: I460V, I460T, I460S, I460L, previously identified from the tested PAL-library by other assays. Our results validate the applicability of AncLAAO for coupled enzyme systems with phenylalanine ammonia-lyases, including cell-based assays suitable for the high-throughput screening of directed evolution-derived PAL-libraries.