Ben Tovim_2026_Protein.Sci_35_e70729

Microorganisms use diverse small-molecule signals to coordinate behavior, supporting interspecies and interkingdom communication. These chemical dialogs shape fundamental biological processes and define interactions from cooperation to antagonism. Quorum-quenching (QQ) enzymes, which degrade or modify quorum sensing (QS) molecules such as N-acyl-homoserine lactones (AHLs), may play a pivotal role in shaping these networks. While bacterial QQ lactonases are well-characterized, fungal lactonases remain undercharacterized, including their potential to target both bacterial QS molecules and fungal secondary metabolites. Using phylogenetic and structural analysis, we identified putative lactonases from the metallo-beta-lactamase-like lactonase family in fungi and biochemically validated the activity of one of them, from Aspergillus pseudonomiae. Structural alignment between bacterial lactonases and the AlphaFold-predicted structure of the fungal homolog further supports that the fungal protein is indeed a lactonase. We then applied Protein Repair One-Stop Shop algorithm for stability engineering to design a variant with improved expression in Escherichia coli and thermal stability. Purified enzyme demonstrated broad substrate specificity, effectively degrading various bacterial AHLs, with k(cat)/K(M) values of up to 1.4 x 10(5) s(-1)M(-1). Both docking analysis and in vitro assays indicated that the enzyme could accommodate and act on the fungal secondary metabolite patulin, a mycotoxin associated with post-harvest disease, showing high catalytic efficiency (1.1 x 10(4) s(-1)M(-1)). When added exogenously to microbial cultures, the enzyme reduced bacterial biofilms and impaired fungal hyphal development, with the hyphal inhibition accompanied by a concentration-dependent increase in ROS. Together, these results show that this fungal enzyme functions as a dual-acting lactonase, positioning it as an interference enzyme in chemical signaling of fungi and bacteria and as a means to suppress pathogens and reduce patulin contamination in food.