An AHL-lactonase mutant featuring a unique "tri-His" motif exhibits enhanced activity, stability and effectively...

Quorum quenching through AHL-lactonase has been established as a critical approach for managing quorum sensing-mediated bacterial infections. While numerous studies have concentrated on enhancing the activity of AHL lactonases, concurrent improvements in both activity and stability have remained elusive. In this study, we adopted a hybrid strategy involving rational and semirational design to concurrently increase the activity and stability of the marine AHL-lactonase AhlX. The mutant M41 (E77I/D157G/T243Y/H255L) exhibited a significant increase in catalytic efficiency, with an 11-fold increase in k_cat/K_m, as well as a substantial increase in thermal stability, with a 12 °C increase in the melting temperature and a 0.6-fold longer half-life at 70 °C relative to those of wild-type AhlX. Structural insights from crystallographic analysis revealed a unique "tri-His" motif within the homohexamer that is pivotal for its stability. Removal of the "tri-His" motif from the homohexamer rendered the H158A mutant prone to thermal oligomer disassembly. Incorporation of the D157G mutation disrupted the D157-R122 salt bridge, stabilizing this motif. The T243Y and H255L mutations modify the active site conformation by reshaping surface interactions, enhancing both enzymatic activity and stability. Biocontrol experiments revealed that M41 was highly effective at suppressing potato soft rot caused by Pectobacterium carotovorum, primarily by inhibiting the swimming motility of the bacterium. This work not only deepens our understanding of the structure-activity relationships of AHL-lactonases but also lays a solid theoretical foundation for the engineering of these enzymes for biocontrol applications.