Dual-Substrate Selectivity and Thermal Stability Mechanisms of a Novel Nicotiana tabacum Phenylalanine Ammonia-Lyase

The nonoxidative deamination of β-branched aromatic compounds, such as phenylalanine, is a key step in the synthesis of natural products and pharmaceutically active ingredients. However, it remains challenging due to the complexity of the substrates. Phenylalanine ammonia-lyase (PAL) is a promising green catalyst, but its application is hindered by the limited availability of suitable types, low catalytic activity, and poorly understood mechanistic properties. Here, we report a novel PAL in Nicotiana tabacum ( Nt PAL). Through comprehensive computational analysis, we elucidated the mechanisms underlying the enhanced thermostability and dual-substrate affinity of Nt PAL, which represents the first PAL with substrate promiscuity identified in dicotyledonous plants. By generating multisite substitution mutants, we identified two parallel mechanisms for thermostability, entropy-driven and enthalpy-driven, and clarified the roles of key amino acids and substrate trajectories in catalytic process. This study provides new insights into the functional mechanisms of Nt PAL and presents an efficient biocatalyst for asymmetric synthesis.