Phenylalanine ammonia lyase (EC 4.3.1.24; PAL) is a non-hydrolytic enzyme which catalyzes the non-oxidative deamination of l-phenylalanine to <i>trans</i>-cinnamic acid and ammonia. This enzyme has widespread application in industry, agriculture and medicine. PAL has gained interest due to its use in treatment for phenylketonuria (PKU), tyrosinemia, and as amino acid deprivation approach for cancer. Considerable research has been focused on improving the catalytic activity of PAL, but the efforts have been far from comprehensive. Though extensive body of research exists on function, structure, and mechanism of PAL, a systematic study exploring the sequence-function space has not been attempted. Previously, we developed a directed evolution strategy to engineer PAL in <i>E. coli</i>. Here, we report detailed mutational landscape of PAL by performing deep DNA sequencing of the libraries obtained after growth enrichment. Based on the deep mutational scanning (DMS) analysis of PAL, we identified 79 mutational hotspots which exhibited positive change in fitness. Using structure-function approach we picked seven sites for comprehensive single or multi-site saturation mutagenesis in an attempt to further improve the catalytic activity of PAL. We observed that while most mutations were deleterious in combinations, few mutants showed improved fitness when present in combination. Using this new DMS-guided library we were able to identify new combination of mutants with 2.5-fold improvement in the catalytic activity. Further, to understand the mechanistic role of these mutants, we performed QM/MM and observed that different mutants contributed in a unique way to improve the catalytic activity. This included i) decreased root mean square fluctuation (RMSF) of substrate in the active site, ii) greater proximity of the substrate to catalytic residues, iii) stabilization of the substrate in the near attack conformation, iv) stabilization of the transition state, and v) improved tunnelling of the substrate to the active site. In summary, this study significantly advances basic and applied enzymology of PALs.

Deep mutational scanning guided directed evolution reveals multiple mechanisms to improve enzyme function

ACS Fall 2021

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