Dual-Substrate Selectivity and Thermal Stability Mechanisms of a Novel <i>Nicotiana tabacum</i> 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 <i>Nicotiana tabacum</i> (<i>Nt</i>PAL). Through comprehensive computational analysis, we elucidated the mechanisms underlying the enhanced thermostability and dual-substrate affinity of <i>Nt</i>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 <i>Nt</i>PAL and presents an efficient biocatalyst for asymmetric synthesis.