Structural basis of enzymatic functional divergence in the PAL gene family of Salix brachista: Allosteric regulation and catalytic activity mediated by non-catalytic sites.

Phenylalanine ammonia-lyase (PAL) is the rate-limiting enzyme of the plant phenylpropanoid pathway, and its functional divergence is closely associated with environmental adaptation. Using the alpine woody plant Salix brachista as a model, we integrated multi-omics and molecular modeling approaches to systematically characterize the evolutionary and enzymatic functional divergence of the SbrPAL gene family. Six SbrPAL members were identified genome-wide; in vitro enzymatic analysis revealed that half (SbrPAL2, SbrPAL5, SbrPAL6) had completely lost catalytic activity. Compared to its lowland relative Salix suchowensis, S. brachista harbors a significantly higher proportion of inactivated PAL members, suggesting it may have been subjected to specific selective pressures in the alpine habitat. Evolutionary analysis confirmed that the SbrPAL family is predominantly constrained by purifying selection, yet multiple positively selected sites were detected across individual members. Population-level resequencing data from 78 natural populations further revealed that high-frequency nonsynonymous mutations and frameshift INDELs causing premature termination constitute the primary genetic basis for functional degeneration. Site-directed mutagenesis successfully restored catalytic activity in all three inactivated members (SbrPAL2 N103Y, SbrPAL5 F251L, SbrPAL6 E101D/T686R), suggesting that variations at non-catalytic sites may contribute to allosteric regulation by altering protein stability and substrate-binding geometry. Molecular dynamics simulations and machine learning analyses indicated that activity loss and recovery correspond to coordinated remodeling of the protein dynamic network and free energy landscape, converging toward a pre-organized, catalytically competent conformational ensemble. This study provides a mechanistic framework for understanding how key metabolic enzymes evolve under extreme environmental conditions.