Novel biodegradation pathway of insecticide flonicamid mediated by an amidase and its unusual substrate spectrum

The insecticide flonicamid (FLO) and its main degradation intermediate 4-trifluoromethylnicotinamide (TFNA-AM) are hazardous to the environment and animals. Microbial transformation of FLO has been well studied, but no study has yet reported on TFNA-AM degradation by a microorganism. Here, Pseudomonas stutzeri CGMCC 22915 effectively degraded TFNA-AM to 5-trifluoromethylnicotinic acid (TFNA). P. stutzeri CGMCC 22915 degraded 60.0% of TFNA-AM (1154.44 μmol/L) within 6 h with a half-life of just 4.5 h. Moreover, P. stutzeri CGMCC 22915 significantly promoted TFNA-AM decomposition in surface water. The reaction was catalyzed by an amidase, PsAmiA. PsAmiA is encoded in a novel nitrile-converting enzyme gene cluster. The enzyme shared only 20-44% identities with previously characterized signature amidases. PsAmiA was successfully expressed in Escherichia coli and its enzymatic properties were investigated using TFNA-AM as the substrate. PsAmiA was more active toward amides without hydrophilic groups, and did not hydrolyze another amide metabolite of FLO, N-(4-trifluoromethylnicotinoyl)glycinamide (TFNG-AM), which is structurally very similar to TFNA-AM. Molecular docking of PsAmiA and TFNA-AM indicated that hydrophobic residues Leu148, Ala150, Ala195, Ile225, Trp341, Leu460, and Ile463 may affect its substrate spectrum. This study provides new insights of the environmental fate of FLO at the molecular level and the structure-function relationships of amidases.