The Metabolism Of Flavin-Containing Monooxygenase On Insecticides And Sinapine

Flavin-containing monooxygenase（FMOs）is a kind of ancient and highly conserved enzyme,which is widely existed in the biological world.In the past,it was believed that FMO is a sister enzyme of cytochrome P450,which mainly plays a detoxification role in organisms and is a metabolic detoxification enzyme.Mammalian FMO enzymes have metabolic effects on many drugs.Insects FMO is also involved in the detoxification metabolism of plant secondary compounds.Previous studies have shown that the high expression of FMO composition type in the drug-resistant populations of Spodoptera exigua in our laboratory.It indicates that FMO is related to the detoxification metabolism of insecticides and the resistance of pests.In addition to detoxification and metabolism,our studies also found that FMO affects physiological processes such as the balance of glucose and lipid metabolism.In order to explore the physiological mechanism of FMO mediated glucose and lipid metabolism,metabolomic techniques were used to find that FMO expression level affected the level of sinapine in Drosophila.In this study,the metabolic effects of insect FMO on sinapine and insecticides and their mechanisms were investigated,and the following results were obtained:1.Metabolic effect of flavin monooxygenase on sinapinePrevious metabolomic analysis showed that with the increase or decrease of FMO expression in Drosophila,the level of sinapine in vivo significantly decreased or increased,suggesting that FMO may be involved in the metabolism of sinpine.In this study,the metabolic effect of prokaryotic expression of FMO2（Se FMO2）on sinapine was analyzed.The results showed that the content of sinapine decreased significantly after Se FMO2 and sinapine were incubated in vitro,which decreased by 83%at 24 h.The metabolite was identified by mass spectrometry as a molecule with a mass to charge ratio of 309.15.However,further studies showed that FMO could not directly oxidize sinapine,but indirectly metabolize sinapine through H₂O₂released by FMO.For the first time,it was found that insect FMO can produce and release H₂O₂,and the production depends on the level of NADPH,while FMO substrate can inhibit the production of H₂O₂.The product of H₂O₂ oxidation of sinapine is also a molecule with a mass charge ratio of 309.15.The structure analysis of the ion fragment of 309.15 by two-stage mass spectrometry shows that this molecule is a dimer formed by the 8-8’polymerization of sinapine under the action of reactive oxygen species.This study revealed the internal reason why the increase of FMO expression level decreased the level of sinapine in Drosophila,while the down-regulation of FMO expression resulted in the accumulation of sinapine,which was that the reactive oxygen species produced by FMO indirectly participated in the metabolism of sinapine.The results of this study demonstrate for the first time that FMO is involved in a series of physiological,biochemical and metabolic processes in vivo through the production of reactive oxygen species.2.Metabolic effect of flavin monooxygenase on insecticidesIn order to analyze the relationship between FMO and insecticide resistance,the effect of FMO overexpression in Drosophila on the sensitivity to cartap and dinotefuran was further determined on the basis of previous studies.It was found that the sensitivity of Dm FMO1 and Dm FMO2 overexpressed Drosophilus was significantly decreased to cartap and dinotefuran.The LC₅₀ of cartap was increased by 2.24 and 1.37 times,respectively,and that of dinotefuran was increased by 1.27 and 1.20 times,respectively.On this basis,combined with the results of previous studies,the metabolic effects of Se FMO2 on insecticides such as cartap,dinotefuran,metaflumizone,indoxacarb,clothianidin,nitenpyram and chlorantraniliprole were analyzed.The results showed that Se FMO2 had no metabolic activity against dinotefuran,metaflumizone,indoxacarb,clothianidin,nitenpyram and chlorantraniliprole,but only had a direct metabolic effect on cartap.The structure analysis of the metabolites showed that the metabolites were firstly transformed into the nereistoxin,and the nereistoxin was N-oxidized under the action of FMO enzyme to form the N-oxide of Nereistoxin（m/z=166.03）.Since FMO can produce physiological functions by releasing H₂O₂,this study also analyzed the metabolic effect of H₂O₂ on insecticides.HPLC analysis showed that the contents of dinotefuran,metaflumizone,indoxacarb,clothianidin and chlorantraniliprole did not decrease after incubation with H₂O₂,but the contents of nitenpyram decreased significantly under the action of H₂O₂,indicating that H₂O₂can directly oxidize nitenpyram.Mass spectrometry analysis showed that H₂O₂ acts on the C=C double bond of nitenpyram to form hydroxylated products and delete nitrogenous groups to form corresponding acids.This is the first report that reactive oxygen species can directly oxidize insecticides.In conclusion,FMO has a direct metabolic effect on the cartap,oxidizing to form the N oxide of nereistoxin,but has no direct oxidation effect on the other insecticides.However,FMO can participate in metabolic processes in vivo through the generation of H₂O₂,such as the metabolism of nitenpyram and the oxidative polymerization of sinapine.The previously reported high expression of FMO mediated insecticide resistance may be an indirect physiological effect caused by the release of H₂O₂ by FMO,and FMO may exert a wide range of effects on the metabolic process of organisms through this characteristic.