Roles of the N-terminal motif in improving the activity and soluble expression of phenylalanine ammonia lyases in Escherichia coli.

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.