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-1 pmol-1 protein 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 Km 5.99 ± 0.95 µm and kcat 0.03 ± 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. CONCLUSION: 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.
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-1 pmol-1 protein 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 Km 5.99 ± 0.95 µm and kcat 0.03 ± 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. CONCLUSION: 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.
Authors: Cassandra L Ettinger; Frank J Byrne; Inaiara de Souza Pacheco; Dylan J Brown; Linda L Walling; Peter W Atkinson; Richard A Redak; Jason E Stajich Journal: BMC Genomics Date: 2022-10-22 Impact factor: 4.547
Authors: Junaid Ali Siddiqui; Muhammad Musa Khan; Bamisope Steve Bamisile; Muhammad Hafeez; Muhammad Qasim; Muhammad Tariq Rasheed; Muhammad Atif Rasheed; Sajjad Ahmad; Muhammad Ibrahim Shahid; Yijuan Xu Journal: Front Microbiol Date: 2022-05-03 Impact factor: 6.064
Authors: Jean-Noël Houchat; Alison Cartereau; Anaïs Le Mauff; Emiliane Taillebois; Steeve H Thany Journal: Int J Environ Res Public Health Date: 2020-05-06 Impact factor: 3.390