Cao_2026_J.Hazard.Mater_510_142095

Pyraclostrobin has emerged as a persistent environmental contaminant across diverse ecosystems, posing potential risks to human health and necessitating effective remediation strategies. In this study, Priestia megaterium GXU087 degraded 98.67% of pyraclostrobin (30 mg/L) within 4 d under optimal conditions. UPLC-HRMS analysis identified two metabolites (M373, M329), leading to the proposal of two biodegradation pathways primarily involving ester bond hydrolysis and C-N bond cleavage. ECOSAR assessment revealed reduced ecotoxicity of these metabolites toward aquatic organisms compared to the parent compound. Through genomic screening and heterologous expression, we identified a novel pyraclostrobin-degrading carboxylesterase, PymH, which represents the first enzyme reported to catalyze the hydrolytic cleavage of the C-N bond in pyraclostrobin. The purified PymH exhibited optimal activity (k(cat)/K(m) of 0.75 min(-1) microM(-1)) at 40 degreesC and pH 8.0 and displayed notable functional stability across broad temperature (20-45 degreesC) and pH (6.0-10.0) ranges, along with tolerance to multiple metal ions (Mn(2+), Fe(2+), Ni(2+), Co(2+), Ca(2+), Mg(2+), 1 mM). Molecular docking and site-directed mutagenesis revealed the catalytic mechanism of PymH, confirming the essential role of the catalytic triad (S93-H222-D192) in the degradation process. Notably, we constructed three mutants (I120A, K121A, I120A-K121A) based on an activity-enhancing mechanism involving accelerated substrate capture and improved binding affinity that increased relative activities to 206.18%, 356.43%, and 411.62% of wild-type PymH, respectively. In summary, this work contributes valuable novel bacterial and enzymatic resources for pyraclostrobin bioremediation and advances the mechanistic understanding of its microbial degradation.