title: Imidacloprid is degraded by CYP353D1v2, a cytochrome P450 overexpressed in a resistant strain of Laodelphax striatellus.
abstract: BACKGROUND
  Cytochrome P450s are associated with the metabolising of a wide range of compounds, including insecticides. CYP353D1v2 has been found to be overexpressed in an imidacloprid-resistant strain of Laodelphax striatellus. Thus, this study was conducted to express CYP353D1v2 in Sf9 cells as a recombinant protein, to assess its ability to metabolise imidacloprid.
  
  
  RESULTS
  Western blot and carbon monoxide difference spectrum analysis indicated that the intact CYP353D1v2 protein had been successfully expressed in Sf9 insect cells. Catalytic activity tests with four traditional P450-activity-probing substrates found that the expressed CYP353D1v2 preferentially metabolised p-nitroanisole, ethoxycoumarin and ethoxyresorufin with specific activities of 32.70, 0.317 and 1.22 pmol min-1pmol-1protein respectively, but no activity to luciferin-H EGE. The enzyme activity for degrading imidacloprid was tested by measuring substrate depletion and formation of the metabolite. Kinetic parameters for imidacloprid were Km5.99 ± 0.95 µm and kcat0.03 ± 0.0004 min-1. The chromatogram analysis showed clearly the NADPH-dependent depletion of imidacloprid and the formation of an unknown metabolite. The UPLC-MS mass spectrum demonstrated that the metabolite was an oxidative product of imidacloprid, 5-hydroxy-imidacloprid.
  
  
  CONCLUSIONS
  These results suggest that CYP353D1v2 in L. striatellus is capable of degrading imidacloprid, and that enzyme activity can be evaluated well only by some traditional probing substrates. © 2017 Society of Chemical Industry.
authors.affiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Mohammed Esmail Abdalla
authors.initials: MEA
authors.lastname: Elzaki
authors.affiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Mohammad Asaduzzaman
authors.initials: MA
authors.lastname: Miah
authors.affiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.affiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Haomiao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Pu
authors.affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Zhaojun
authors.initials: Z
authors.lastname: Han
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006430:Hemiptera / Q000201:enzymology* / Q000235:genetics; D008658:Inactivation, Metabolic / Q000235:genetics; D019476:Insect Proteins / Q000378:metabolism; D007305:Insecticide Resistance; D007306:Insecticides / Q000378:metabolism; D000073943:Neonicotinoids / Q000378:metabolism*; D009574:Nitro Compounds / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D019476:Insect Proteins; D007306:Insecticides; D000073943:Neonicotinoids; D009574:Nitro Compounds; C082359:imidacloprid; D003577:Cytochrome P-450 Enzyme System
keywords: cytochrome P450; functional expression; imidacloprid detoxification; insecticide metabolism; probing substrates