Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides

Cheesman, Matthew J., Traylor, Matthew. J., Hilton, Margaret E., Richards, Katelyn E., Taylor, Matthew C., Daborn, Philip J., Russell, Robyn J., Gillam, Elizabeth M. J. and Oakeshott, John G. (2013) Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides. Insect Biochemistry and Molecular Biology, 43 5: 455-465. doi:10.1016/j.ibmb.2013.02.003


Author Cheesman, Matthew J.
Traylor, Matthew. J.
Hilton, Margaret E.
Richards, Katelyn E.
Taylor, Matthew C.
Daborn, Philip J.
Russell, Robyn J.
Gillam, Elizabeth M. J.
Oakeshott, John G.
Title Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides
Formatted title
Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides
Journal name Insect Biochemistry and Molecular Biology   Check publisher's open access policy
ISSN 0965-1748
1879-0240
Publication date 2013-05-01
Year available 2013
Sub-type Article (original research)
DOI 10.1016/j.ibmb.2013.02.003
Open Access Status Not Open Access
Volume 43
Issue 5
Start page 455
End page 465
Total pages 11
Place of publication Kidlington, Oxford, United Kingdom
Publisher Pergamon
Language eng
Abstract The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b5 enhanced the kcat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min(-1)) with little effect on KM (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.
Formatted abstract
Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L-1 of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.
Keyword Cytochrome P450
CYP6G1
Pesticide
Multi-pesticide resistance
Insecticide resistance
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
School of Chemistry and Molecular Biosciences
 
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