Integrated transcriptomic and enzymatic characterization of the molecular insights into nicosulfuron biodegradation by Enterobacter ludwigii ES2.

Nicosulfuron residues pose a threat to agricultural ecosystems. Therefore, it is crucial to explore the molecular mechanisms underlying microbial degradation for effective remediation. This study elucidated the nicosulfuron degradation pathway by Enterobacter ludwigii ES2 and identified a key enzyme involved in its detoxification. UPLC-QTOF-HRMS-MS analysis revealed two major degradation products, 2-amino-sulfonyl-N,N-dimethylnicotinamide and 2-amino-4,6-dimethoxypyrimidine, formed via deamidation and pyrimidine ring demethylation. Transcriptomic analysis indicated differentially expressed genes in response to nicosulfuron, suggesting candidates associated with nicosulfuron degradation. Gene knockout and complementation experiments confirmed that disruption of El-rutA reduced its degradation efficiency by ∼60 %, verifying its essential role. Heterologously expressed and purified El-RutA (39.90 kDa) exhibited optimal activity (>90 %) at 30 °C, pH 7.0, and 50 mg L nicosulfuron, with Zn or Mg further enhancing activity. Substrate specificity assays showed that El-RutA catalyzed the demethylation of 2-amino-4,6-dimethoxypyrimidine. Molecular docking and dynamics simulations revealed a strong binding affinity (-8.827 kcal/mol) between El-RutA and nicosulfuron, stabilized by hydrogen bonds, with Lys-227 identified as a critical residue for substrate interaction via mutagenesis. An engineered Bacillus subtilis WB600[pWB980-El-rutA] strain degraded 60.10 % of nicosulfuron, and soil bioremediation tests demonstrated its practical applicability. This study provides the first mechanistic insight into El-RutA-mediated nicosulfuron degradation and demonstrates its utility in an engineered strain for bioremediation, thereby offering a basis for microbial strategies to mitigate nicosulfuron contamination.