Unraveling the role of PxGSTD5 in pyrethroids metabolism: a molecular and biochemical investigation of resistance mechanisms in Plutella xylostella

Abstract Plutella xylostella , a major pest of cruciferous crops, has rapidly evolved resistance to pyrethroid insecticides, posing a significant threat to sustainable agriculture. Among the various resistance mechanisms, glutathione S‐transferases (GSTs) play a pivotal role in the detoxification of xenobiotics. However, the molecular basis underlying their interaction with pyrethroids remains poorly understood. In this study, we investigated the metabolic function of PxGSTD5, a Delta‐class GST from P. xylostella , in the detoxification of two widely used pyrethroids‐esfenvalerate and deltamethrin‐through a comprehensive approach integrating enzyme kinetics, in vitro metabolism assays, molecular docking, and site‐directed mutagenesis. Our results revealed that PxGSTD5 exhibited substantial metabolic activity toward esfenvalerate (31.65%) and deltamethrin (41.20%). Molecular docking and mutagenesis analyses identified Ser12, His41, Tyr116, and Phe120 as key amino acid residues responsible for substrate binding and catalysis. Alanine substitution of these residues significantly impaired the enzyme's metabolic efficiency, underscoring their critical roles in GST‐mediated detoxification. This study provides direct biochemical and structural evidence for the involvement of PxGSTD5 in pyrethroid metabolism and resistance in P. xylostella . These findings offer valuable insights into the molecular mechanisms of detoxification and support the rational design of next‐generation insecticides capable of overcoming GST‐mediated resistance, thereby contributing to improved pest management strategies.