Revealing EPS1's Catalytic Mechanism in the Biosynthesis of Salicylic Acid via Computer Modeling

EPS1, a member of the BAHD acyltransferase protein family, functions as an isochorismoyl-glutamate pyruvoyl-glutamate lyase (IPGL) that plays a pivotal role in the conversion of isochorismate to salicylic acid (SA) in Brassicaceae plants. The elucidation of EPS1's specific catalytic process for SA synthesis has presented challenges, primarily due to the lack of structural and dynamic insights into the interaction between EPS1 and its natural substrate, isochorismoyl-glutamate A (IGA). In this study, we employed molecular docking, simulated annealing, and MD simulations to predict the binding sites of IGA and IPGL products. Subsequently, utilizing the modeled holo structure, we conducted in silico mutations to unveil the distinct functions of critical residues that have been experimentally proven to disrupt IPGL activity. Our comparison of the substrate and IPGL products in their free and bound states revealed a unique pericyclic rearrangement lyase mechanism facilitated by EPS1's active site, leading to SA production. Through mutant simulations analysis, we further demonstrated the essential protective role of Thr306, a residue intermittently involved in substrate binding, in maintaining IPGL activity. Overall, our research provides comprehensive insights into the catalytic mechanism of EPS1 in SA biosynthesis, offering valuable knowledge on the complex pathways of plant SA metabolism and potential strategies for enhancing crop disease resistance through innovative engineering methods.