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.

Characterization of nitrilases from Variovorax boronicumulans that functions in insecticide flonicamid degradation and ß-cyano-L-alanine detoxification.

AIMS: To characterize the functions of nitrilases of Variovorax boronicumulans CGMCC 4969 and evaluate flonicamid (FLO) degradation and ß-cyano-L-alanine (Ala(CN)) detoxification by this bacterium. METHODS AND RESULTS: Variovorax boronicumulans CGMCC 4969 nitrilases (NitA and NitB) were purified, and substrate specificity assay indicated that both of them degraded insecticide FLO to N-(4-trifluoromethylnicotinoyl)glycinamide (TFNG-AM) and 4-(trifluoromethyl)nicotinol glycine (TFNG). Ala(CN), a plant detoxification intermediate, was hydrolysed by NitB. Escherichia coli overexpressing NitA and NitB degraded 41.2 and 93.8% of FLO (0.87 mmol·L-1 ) within 1 h, with half-lives of 1.30 and 0.25 h, respectively. NitB exhibited the highest nitrilase activity towards FLO. FLO was used as a substrate to compare their enzymatic properties. NitB was more tolerant to acidic conditions and organic solvents than NitA. Conversely, NitA was more tolerant to metal ions than NitB. CGMCC 4969 facilitated FLO degradation in soil and surface water and utilized Ala(CN) as a sole nitrogen source for growth. CONCLUSIONS: CGMCC 4969 efficiently degraded FLO mediated by NitA and NitB; NitB was involved in Ala(CN) detoxification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study promotes our understanding of versatile functions of nitrilases from CGMCC 4969 that is promising for environmental remediation.

Nitroreduction of imidacloprid by the actinomycete Gordonia alkanivorans and the stability and acute toxicity of the nitroso metabolite.

The insecticide imidacloprid (IMI), which is used worldwide, pollutes environments and has significant ecotoxicological effects. Microbial metabolism and photolysis are the major pathways of IMI degradation in natural environments. Several studies have reported that the metabolites of IMI nitroreduction are more toxic to some insects and mammals than IMI itself. Thus, environmental degradation of IMI may enhance the ecotoxicity of IMI and have adverse effects on non-target organisms. Here, we report that an actinomycete-Gordonia alkanivorans CGMCC 21704-transforms IMI to a nitroreduction metabolite, nitroso IMI. Resting cells of G. alkanivorans at OD600 nm = 10 transformed 95.7% of 200 mg L-1 IMI to nitroso IMI in 4 d. Nitroso IMI was stable at pH 4-9. However, it rapidly degraded under sunlight via multiple oxidation, dehalogenation, and oxidative cleavage reactions to form 10 derivatives; the half-life of nitroso IMI in photolysis was 0.41 h, compared with 6.19 h for IMI. Acute toxicity studies showed that the half maximal effective concentration (EC50) values of IMI, nitroso IMI, and its photolytic metabolites toward the planktonic crustacean Daphnia magna for immobilization (exposed to the test compounds for 48 h) were 17.70, 9.38, 8.44 mg L-1, respectively. The half-life of nitroso IMI in various soils was also examined. The present study reveals that microbial nitroreduction accelerates IMI degradation and the nitroso IMI is easily decomposed by sunlight and in soil. However, nitroso IMI and its photolytic products have higher toxicity toward D. magna than the parent compound IMI, and therefore increase the ecotoxicity of IMI.

Biodegradation of flonicamid by Ensifer adhaerens CGMCC 6315 and enzymatic characterization of the nitrile hydratases involved.

BACKGROUND: Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinamide insecticide that regulates insect growth. Because of its wide application in agricultural production and high solubility in water, it poses potential risks to aquatic environments and food chain. RESULTS: In the present study, Ensifer adhaerens CGMCC 6315 was shown to efficiently transform FLO into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) via a hydration pathway mediated by two nitrile hydratases, PnhA and CnhA. In pure culture, resting cells of E. adhaerens CGMCC 6315 degraded 92% of 0.87 mmol/L FLO within 24 h at 30 °C (half-life 7.4 h). Both free and immobilized (by gel beads, using calcium alginate as a carrier) E. adhaerens CGMCC 6315 cells effectively degraded FLO in surface water. PnhA has, to our knowledge, the highest reported degradation activity toward FLO, Vmax = 88.7 U/mg (Km = 2.96 mmol/L). Addition of copper ions could increase the enzyme activity of CnhA toward FLO by 4.2-fold. Structural homology modeling indicated that residue ß-Glu56 may be important for the observed significant difference in enzyme activity between PnhA and CnhA. CONCLUSIONS: Application of E. adhaerens may be a good strategy for bioremediation of FLO in surface water. This work furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and provides effective transformation strategies for microbial remediation of FLO contamination.

Biodegradation of the pyridinecarboxamide insecticide flonicamid by Microvirga flocculans and characterization of two novel amidases involved.

Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinecarboxamide insecticide that exhibits particularly good efficacy in pest control. However, the extensive use of FLO in agricultural production poses environmental risks. Hence, its environmental behavior and degradation mechanism have received increasing attention. Microvirga flocculans CGMCC 1.16731 rapidly degrades FLO to produce the intermediate N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) and the end acid metabolite 4-(trifluoromethyl) nicotinol glycine (TFNG). This bioconversion is mediated by the nitrile hydratase/amidase system; however, the amidase that is responsible for the conversion of TFNG-AM to TFNG has not yet been reported. Here, gene cloning, overexpression in Escherichia coli and characterization of pure enzymes showed that two amidases-AmiA and AmiB-hydrolyzed TFNG-AM to TFNG. AmiA and AmiB showed only 20-30% identity to experimentally characterized amidase signature family members, and represent novel amidases. Compared with AmiA, AmiB was more sensitive to silver and copper ions but more resistant to organic solvents. Both enzymes demonstrated good pH tolerance and exhibited broad amide substrate specificity. Homology modeling suggested that residues Asp191 and Ser195 may strongly affect the catalytic activity of AmiA and AmiB, respectively. The present study furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and may aid in the development of a bioremediation agent for FLO.