Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

Accepted Manuscript Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides Matthew J. Traylor, Jong-Min Baek, Katelyn E. Richards, Roberto Fusetto, W. Huang, Peter Josh, Zhengzhong Chen, Padma Bollapragada, Richard A.J. O'Hair, Philip Batterham, Elizabeth M.J. Gillam PII:

S0965-1748(17)30136-4

DOI:

10.1016/j.ibmb.2017.09.004

Reference:

IB 2990

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 4 August 2017 Revised Date:

8 September 2017

Accepted Date: 10 September 2017

Please cite this article as: Traylor, M.J., Baek, J.-M., Richards, K.E., Fusetto, R., Huang, W., Josh, P., Chen, Z., Bollapragada, P., O'Hair, R.A.J., Batterham, P., Gillam, E.M.J., Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Recombinant expression and characterization of Lucilia cuprina CYP6G3:

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activity and binding properties toward multiple pesticides

3 Matthew J. Traylora, Jong-Min Baeka, Katelyn E. Richardsa, Roberto Fusettob, W. Huang,

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Peter Josha, Zhengzhong Chenb, Padma Bollapragadaa, Richard A. J. O’Hairb, Philip

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Batterhamb, Elizabeth M.J. Gillama*

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Australia

School of Chemistry and Molecular Biology, University of Queensland, St. Lucia 4072,

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The Bio21 Institute, The University of Melbourne, Parkville, Victoria, 3010, Australia,

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*Corresponding author. Tel.: +61 7 3365 1410; Fax.: +61 7 3365 4699; E-mail address:

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[email protected]

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Abstract

15 The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a

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major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of

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L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue

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of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To

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investigate the basis of resistance, a bicistronic Escherichia coli expression system was

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developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450

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reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening

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substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-

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benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The

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addition of house fly cytochrome b5 enhanced the kcat for p-nitroanisole dealkylation

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approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min-1) with little effect on KM (13 ± 1 vs 10 ±

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1 µM). Inhibition studies and difference spectroscopy revealed that the organochlorine

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compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and

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chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding

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spectra with spectral dissociation constants in the micromolar range suggesting that they may

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be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate,

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diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral

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assays, with a Kd value of 23 +/- 3 µM. CYP6G3 metabolised diazinon to the diazoxon and

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hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites,

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consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and

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neonicotinoid insecticides in other species.

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Keywords: cytochrome P450, CYP6G3, Lucilia cuprina, Australian Sheep Blowfly,

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insecticide resistance, diazinon, imidacloprid

40 Abbreviations: 7EC – 7-ethoxycoumarin; BR- 7-benzyloxyresorufin; CuOOH – cumene

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hydroperoxide; δ-ALA – delta-aminolevulinic acid; DDT – dichlorodiphenyltrichloroethane

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DTT – dithiothreitol; EDTA - ethylenediaminetetraacetic acid; fCPR – cytochrome P450

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reductase from Musca domestica; fb5 – cytochrome b5 from Musca domestica; IPTG -

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isopropyl β-D-1-thiogalactopyranoside; IMP - 2-isopropyl-6-methyl-4-pyrimidinol; LC-

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MS/MS – liquid chromatography mass spectrometry/mass spectrometry; luciferin-BE -

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luciferin 6’-benzyl ether; luciferin-CEE - luciferin 6’-chloroethyl ether; luciferin-H - 6’-

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deoxyluciferin; luciferin-H-EGE - 6’-deoxyluciferin 4-ethylene glycol ester; luciferin-ME -

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luciferin 6’-methyl ether; luciferin-ME-EGE - luciferin 6’-methyl ether 4-ethylene glycol

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ester; luciferin-PFBE - luciferin 6’-pentafluorobenzyl ether; P450 – cytochrome P450; PMSF

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- phenylmethylsulfonyl fluoride; pNAOD – p-nitroanisole O-dealkylation; pNP – p-

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nitrophenol; pNA – p-nitroanisole.

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1. Introduction

55 Cutaneous myiasis, known also as flystrike, is a lethal ectoparasitic infection of sheep.

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Flystrike is most prevalent in Australia—owing to climate, farming conditions and susceptible

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breeds—and costs graziers upwards of AUD150 million annually (Capinera, 2008; Levot,

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2012; Phillips, 2009). Lucilia cuprina, the Australian sheep blowfly, causes 90% of these

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flystrike infections (Levot, 1995b, a; Phillips, 2009). Instances of flystrike are seen globally,

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with warmer tropical regions, such as New Zealand and South Africa, being affected by L.

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cuprina-mediated myiasis more frequently than colder regions, including parts of northern

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Europe, where flystrike is mostly caused by Lucilia sericata (Wall, 2012).

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Traditionally, the process of mulesing, which involves surgically removing skin folds from areas of the sheep where the flies most commonly oviposit, is used as an effective

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method to prevent strike infections. However, mulesing has raised ethical concerns and is

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now illegal in many countries (Phillips, 2009). Chemical methods of flystrike treatment have

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since been applied, with organochlorine, organophosphorus, and triazine pesticides all having

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been used (Joshua and Turnbull, 2015). However, resistance has arisen in L. cuprina to to

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organochlorine, organophosphorus, benzoylurea, pyrethroid, and carbamate pesticides

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(Hughes, 1982; Hughes and Devonshire, 1982; Kotze, 1993; Kotze, 1995; Kotze and Sales,

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1994), further complicating the management of flystrike in the grazing industry (Heath and

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Levot, 2015; Levot, 1995b, a; Phillips, 2009).

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Cytochrome P450 enzymes are a ubiquitous class of hemoproteins present in all

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classes of life. These enzymes catalyse over 60 types of reactions and are responsible for a

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diverse array of biosynthetic and degradative processes in vivo, including xenobiotic

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detoxification (Guengerich, 2001). Many P450 enzymes have been linked to insecticide

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resistance (Feyereisen, 2012; Gillam and Hunter, 2007; Heckel, 2010; Li et al., 2007b). It is

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pesticides and accelerating their clearance from the insect. However, P450s may be associated

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with the metabolic bioactivation of certain pesticides to toxic, active metabolites. An example

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is the conversion of the phosphorothioate, diazinon, to its active metabolite, diazoxon

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(Feyereisen, 2012). P450s have been proposed to both bioactivate diazinon to diazoxon and

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its hydroxyl metabolite and to detoxify it to hydroxydiazinon and 2-isopropyl-6-methyl-4-

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pyrimidinol (IMP) (Ellison et al., 2012; PisaniBorg et al., 1996; Shishido et al., 1972; Wilson

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et al., 1999). An esterase is known to detoxify diazoxon in L. cuprina (Hartley et al., 2006;

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Newcomb et al., 1997) but the enzymes responsible for the metabolism of diazinon to

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diazoxon or hydroxydiazinon are yet to be identified.

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Pesticide resistance in the model fly species Drosophila melanogaster has been extensively characterized (Feyereisen, 2012). In Drosophila, CYP6G1 has been associated

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with resistance to many other classes of insecticides (Cheesman et al., 2013; Daborn et al.,

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2007; Feyereisen, 2012) including the neonicotinoid, imidacloprid (Fusetto et al., 2017; Hoi et

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al., 2014). The proposed CYP6G1 ortholog in houseflies, CYP6G4 has similarly been

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associated with resistance to multiple pesticides, including imidacloprid and spinosad (Gao et

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al., 2012; Hojland et al., 2014).

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P450s have been implicated in L. cuprina resistance for decades (Hughes, 1982; Hughes and Devonshire, 1982; Kotze and Sales, 1994; Kotze et al., 1997) but no biochemical

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studies of specific L. cuprina P450 enzymes have been done to date. Since CYP6G3 from L.

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cuprina is in the same subfamily as CYP6G1 and CYP6G4 we hypothesized that it may also

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play a role in insecticide metabolism. The objective of this work was to develop a bacterial

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expression system to generate active CYP6G3 and to characterise the interaction of this

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enzyme with several classes of insecticides with the aim of gaining a better understanding of

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the xenobiotic detoxification capacities of L. cuprina. In particular, we sought to determine

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the role of CYP6G3 in the metabolism of diazinon and imidacloprid.

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2. Materials and Methods

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107 2.1. Materials

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Isopropyl β-D-1-thiogalactopyranoside (IPTG), δ-aminolevulinic acid (δ-ALA ), DTT,

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bestatin, aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) were

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obtained from Sigma Chemical Co (St. Louis, MO). T4 DNA ligase, was from Promega

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(Madison, WI), and DH5α F′IQ™ Max Efficiency cells were purchased from Invitrogen

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(Carlsbad, CA). Pwo DNA polymerase was from Roche Applied Science (Indianapolis, IN),

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and QIAquick PCR Purification and Gel Extraction Kits were from Qiagen (Valencia, CA).

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P450-glo reagents were from Promega (Madison, WI). The pesticides chlorfenvinphos,

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endosulfan and dicyclanil were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,

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CA), diazinon was obtained from Chem Service (West Chester, PA), and malathion,

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lufenuron, carbaryl, and DDT were obtained from Sigma-Aldrich (St. Louis, MO).

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Imidacloprid (N-[1-[(6-Chloro-3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl]nitramide) was

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obtained from AnalaR (supplied by BDH, Poole, Great Britain). 13C6-imidacloprid was

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provided by IsoSciences (PA, USA). 5-hydroxy-imidacloprid and olefin-imidacloprid were

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purchased from Bayer AG (Leverkusen, North Rhine-Westphalia, Germany). HPLC grade

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acetonitrile and HPLC grade methanol were obtained from Merck (Kenilworth, NJ, U.S.A.).

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Glacial formic acid was provided by AnalaR (supplied by BDH, Poole, Great Britain). 18.2

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MΩ HPLC grade water was obtained from Honeywell (Morristown, NJ, USA). Other

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ACCEPTED MANUSCRIPT chemicals and reagents were of the highest quality commercially available from local

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suppliers and were used as purchased.

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The pCW6G1-Barnes/fCPR vector was from another study (Cheesman et al., 2013). The

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expression vectors for house fly cytochrome b5 and CPR, pCb5 (Guzov et al., 1996) and pI-95

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(Andersen et al., 1994), were generous gifts of Professor R. Feyereisen, while the chaperone

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plasmid set was obtained as a generous gift from Professor K. Nishihara (HSP Research

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Institute, Kyoto Japan) (Nishihara et al., 1998).

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2.2. Construction of the pCW6G3-Barnes/fCPR expression plasmid

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The cDNA for CYP6G3 (Genbank accession number: DQ917668) was obtained from a

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cDNA library as described previously (Lee et al., 2011) and cloned into the pUAS-attB

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vector. A bacterial expression plasmid containing the cDNAs for CYP6G3 and housefly

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cytochrome P450 reductase (fCPR) arranged in a bicistronic format was constructed using the

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pCW6G1-Barnes/fCPR vector (Cheesman et al., 2013) from which the 6G1 cDNA had been

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removed with an NdeI and SalI digestion. The Cyp6g3 gene was amplified from the source

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plasmid, pUAS-attb-Lc6G3, with a forward primer (5’-

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GGAGGTGGACATATGGCTCTGTTATTAGCAGTTTTTTTCATTACCTGTATTCTTGG-

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3’) that incorporated an NdeI restriction site into the start codon and encoded the

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MALLLAVFL N-terminal sequence to facilitate expression (Barnes et al., 1991). The reverse

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primer (5’-

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GTGGTGGTGGTCGACTTAACAAGTATATTTTTATCATAGAGATTATCTCTTATCAC

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-3’) added a SalI restriction site to the end of the open reading frame to enable subcloning into

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the NdeI and SalI sites of the pCW6G1-Barnes/fCPR vector in place of the CYP6G1

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sequence. In this manner, the CYP6G3 sequence was also modified to contain a C-terminal

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hexa-histidine affinity tag connected with a serine/threonine linker to facilitate future

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purification.

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2.3. Expression in E. coli and preparation of bacterial membranes

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The pCW6G3-Barnes/fCPR plasmid was co-expressed in DH5αF’IQTM E. coli cells containing the chaperone expression plasmid pGro7 (Nishihara et al., 1998) using a standard

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expression protocol (Notley et al., 2002). A single colony was inoculated into 5 ml LB media

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with 100 µg/ml ampicillin and 20 µg/ml chloramphenicol and grown overnight with shaking

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at 37°C. Expression cultures of 100 ml terrific broth containing 1 mM thiamine, 100 µg/ml

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ampicillin, 20 µg/ml chloramphenicol, and trace elements (Bauer and Shiloach, 1974) were

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inoculated with 1 ml of starter culture. Expression cultures were incubated at 25°C with

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shaking for 5 h before induction with the addition of 4 mg/ml arabinose, 1 mM IPTG, and 0.5

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mM δ-ALA. Expression cultures were incubated with shaking at 25°C for a further 43 h (48 h

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total) before harvest. Bacterial membranes containing recombinant CYP6G3 and fCPR were

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prepared via ultracentrifugation following lysis in a French Press as described previously

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(Cheesman et al., 2013) and stored at -80°C prior to use.

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To supplement enzymatic reactions with controlled and reproducible amounts of P450

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reductase, membranes containing fCPR were generated from bacteria expressing recombinant

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fCPR alone from the pCW/fCPR vector (Cheesman et al., 2013) in the same manner as

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described above. Additionally, membranes containing housefly cytochrome b5 (fb5) were

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generated similarly from cultures expressing the pCb5 vector (Cheesman et al., 2013; Guzov

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et al., 1996).

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The concentration of CYP6G3 was measured in membrane preparations via Fe (II).CO vs Fe(II) difference spectroscopy (Omura and Sato, 1964). Microsomal fCPR content was

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ACCEPTED MANUSCRIPT measured using cyt c as a surrogate electron acceptor and assuming a specific activity of 3024

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nmol cyt c reduced min-1 nmol fCPR-1(Murataliev et al., 1999). Microsomal fb5 content was

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measured via difference spectroscopy as described by Estabrook and Werringloer (Estabrook

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and Werringloer, 1978) with an OLIS-modified Aminco DW2a spectrophotometer.

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2.4. Characterization of activity toward marker substrates

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Thirteen potential marker substrates were screened for CYP6G3 activity. The

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fluorogenic substrates coumarin (Waxman and Chang, 2006a), 7-ethoxycoumarin (7EC)

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(Waxman and Chang, 2006b) and 7-benzyloxyresorufin (Stresser et al., 2002); the

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colorimetric substrates p-nitrophenol (pNP) (Chang et al., 2006) and p-nitroanisole (pNA)

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(Hansen and Hodgson, 1971); and the luminogenic P450-glo substrates (Cali et al., 2006),

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luciferin-ME, -CEE, -H, –BE, -PFBE, -ME-EGE and –H-EGE were assayed as described

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previously (Cheesman et al., 2013). All reactions included 50 nM CYP6G3 and were initiated

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by the addition of either 500 µM cumene hydroperoxide (CuOOH) for the fluorogenic

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substrates, or an NADPH-regenerating system (10 mM glucose-6-phosphate, 250 µM

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NADP+, and 0.5 U/ml glucose-6-phosphate dehydrogenase) for the colorimetric and

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luminogenic substrates. CuOOH was used as the electron source in the fluorescence assays

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rather than NADPH so that the generation of the dealkylated products could be monitored via

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fluorescence intensity measurements in real time without interference from NADPH

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fluorescence. The electron transfer proteins fCPR and fb5 were supplemented where indicated

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to a final level of 250 nM and 500 nM, respectively, in reactions supported by the NADPH-

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regenerating system.

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Catalytic constants were measured for p-nitroanisole O-dealkylation (pNAOD) in the presence and absence of 500 nM fb5 as described previously (Cheesman et al., 2013).

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ACCEPTED MANUSCRIPT Catalytic constants were measured for 7-ethoxycoumarin O-dealkylation (7ECOD) supported

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by CuOOH in the absence of fb5 or additional fCPR above the level already present in the

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bicistronic membranes. The inhibition of 7ECOD at 1 mM 7EC was measured similarly

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without additional fCPR or fb5 in the presence of 100, 10, 1, 0.1, 0.01 and 0 µM inhibitor.

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GraphPad prism was used to calculate IC50 values using the following equation,

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Y=100/(1+10^((LogIC50-X)*HillSlope)).

208 2.5. Ligand binding experiments

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Ligand-induced difference spectra were recorded in an OLIS-modified Aminco DW2a

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spectrophotometer as previously described (Jefcoate, 1978). CYP6G3-containing membranes

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from cells transformed with the bicistronic vector were diluted to 0.46 µM in TES buffer (50

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mM Tris acetate, 250 mM sucrose, 0.25 mM EDTA, pH 7.6) and the membrane suspension

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was split between sample and reference cuvettes. Ligands were dissolved in methanol and

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sequential, equal additions of dissolved ligand and methanol were added to the sample and

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reference cuvettes respectively, to produce the ligand concentrations indicated in individual

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figures. The total, final concentration of methanol was kept below 2 %.

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2.6. Characterization of imidacloprid metabolism CYP6G3-mediated imidacloprid metabolism was assessed in vitro by incubating

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bacterial membranes with 12C6 and 13C6- imidacloprid in a 50:50 ratio to allow the

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identification of imidacloprid metabolites through the use of the twin-ion method described

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previously (Hoi et al., 2014). The two isotopes of imidacloprid were dissolved in HPLC grade

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water in separate volumetric flasks and the isotopes united only at the start of the experiment.

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Bacterial membranes containing CYP6G3 (0.10 µM) and fCPR (0.18 µM) were incubated

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ACCEPTED MANUSCRIPT with 25 µM or 500 µM imidacloprid (total concentration) in 100 mM potassium phosphate

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buffer, pH 7.4. Reactions were initiated by the addition of an NADPH-regenerating system

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(10 mM glucose-6-phosphate, 250 µM NADP+, and 0.5 U/ml glucose-6-phosphate

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dehydrogenase) as described above. Negative controls lacked either Cyp6g3 (CPR only) or

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the NADPH-generating system components (-NGS) or were terminated immediately after

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addition of the NADPH-generating system (T = 0). A substrate only control was also used

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which comprised substrate added to a buffer solution (Substrate only) to monitor any

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chemical degradation of imidacloprid. After 2 hours at 37°C, the reactions were stopped

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adding 500 µL of cold methanol and transferring the tubes to ice for an hour to facilitate

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precipitation of proteins. The samples were clarified by centrifuging the tubes for 6 minutes at

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16 000 x g and the supernatants were recovered. A second extraction was performed with 500

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µL of cold methanol and the combined extracts were evaporated under vacuum. The dried

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residues were stored in the freezer (-20°C) prior to analysis of the metabolites.

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Imidacloprid metabolites were analysed as described previously by Fusetto et al., (2017).

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Briefly, the dried extracts were resuspended in HPLC grade water, and analysed using an

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Agilent 1100 HPLC autosampler system with a reverse-phase C18 column (2.6 µm, 3.0 × 100

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mm, Kinetex XB-C18, Phenomenex, Inc., Torrance, California, USA) coupled to an Agilent

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6520 Q-TOF mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA).

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Separation was achieved using a two solvent gradient consisting of 100% water (Phase 1) and

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a 70:30:0.1% mixture of acetonitrile, H2O, and formic acid, respectively (Phase 2).

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Imidacloprid, and its 5-hydroxyimidacloprid (5OH) and imidacloprid olefin (Olefin)

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metabolites were analysed in both positive and negative mode and quantified using calibration

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curves (R2=0.989, R2=0.999 and R2=0.999 respectively) generated by multiple injections of

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the pure standard at different concentrations.

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2.7. Characterization of diazinon metabolism Incubations were undertaken as described above with 100 µM diazinon, 0.5 µM CYP6G3 and 0.75 µM fCPR. Reactions were stopped after 2 h with two volumes of

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acetonitrile. Diazinon metabolism was analysed as described in detail previously (Ellison et

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al., 2012) except that an Agilent Zorbax 5µm 4.6 mm x 150 mm SB-C18 column was used.

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LC-MS analysis was performed using a Bruker micrOTOF-Q mass spectrometer operated in

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positive ion mode. Source parameters included a capillary voltage of 4500 V; drying gas

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temperature at 250° C; drying gas flow at 6 L/min; nebuliser pressure 4 bar and collision

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energy of 16 eV. Data was acquired in a data dependent manner with MS2 analyses being

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performed on the three most intense ions. Chromatographic separation was performed using a

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Dionex Ultimate 3000 HPLC system equipped with a Zorbax Eclipse XDB-C-18 column (2.1

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x 50 mm, 3.5 µm) at 300 µL per minute. Solvent A was 2% acetonitrile and solvent B was

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90% acetonitrile. The separation gradient was 0-11 minutes 2% B; 11-14 minutes 45% B; 14-

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16 minutes 95% B. Compound identification was confirmed by matching MS2 fragmentation

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data against the MassBank database (http://www.massbank.jp) and data previously published

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(Kouloumbos et al., 2003).

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3.1. CYP6G3 expression, membrane preparation, and protein quantitation

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The bicistronic expression vector pCW6G3-Barnes/fCPR was generated with the

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cDNA for Cyp6g3 and housefly cytochrome P450 reductase (fCPR). The Cyp6g3 gene was

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modified with the N-terminal MALLLAVFL sequence (Barnes et al., 1991) to facilitate

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ACCEPTED MANUSCRIPT expression in E. coli. Expression yields from the pCW6G3-Barnes/fCPR vector were

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approximately 200 nmol P450 holoprotein per L culture, which yielded membrane

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preparations containing 2.3 µM CYP6G3 holoprotein and 3.2 µM fCPR. A reduced CO-

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bound difference spectrum of CYP6G3-containing membranes is shown in Figure 1. The

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Soret maximum is at 450 nm. The shoulder at 417-420 nm may indicate either some

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conversion of P450 holoprotein to the inactive P420 form or the presence of bacterial

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cytochromes (Kita et al., 1984).

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3.2. Metabolism of marker substrates by CYP6G3

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Thirteen marker substrates were tested for CYP6G3 activity. CYP6G3 showed activity towards the fluorogenic substrate 7-ethoxycoumarin and the colorimetric substrate p-

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nitroanisole, but no significant activity was seen towards p-nitrophenol, coumarin, 7-

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benzyloxyresorufin, or with several luminogenic P450-Glo substrates: luciferin-Me, -CEE, -

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H, –BE, -PFBE, -ME-EGE or –H-EGE.

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The rates of CYP6G3-mediated pNAOD and 7ECOD activity were fit well by the Michaelis-Menten equation over the concentration ranges shown in Figure 2. Kinetic

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constants are shown in Table 1. CYP6G3 pNAOD activity was stimulated by fb5 with a nearly

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two-fold increase in kcat and essentially no change in KM (Figure 2).

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3.3. Screening for inhibition of 7ECOD

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CYP6G3-mediated 7ECOD activity was used to screen a selection of ligands for

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interaction with the CYP6G3 active site, including pesticides, and ketoconazole, a P450

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inhibitor (Figure 3). Ligands were tested for inhibition of 7ECOD at 1 mM 7EC (the

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ACCEPTED MANUSCRIPT measured KM). Chlorfenvinphos, malathion, DDT, endosulfan, and ketoconazole all showed

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significant inhibition of 7ECOD with calculated IC50 values ranging from 0.23 µM to 26.8

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µM (Table 2), while imidacloprid, dicyclanil, lauric acid, diazinon and biphenyl showed

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negligible inhibition at concentrations up to 100 µM. Lufenuron and carbaryl showed slight

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inhibition (data not shown), but the IC50 value exceeded the highest assayed concentration and

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could not be estimated accurately.

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308 3.4. Ligand-binding difference spectra

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Ligand-binding difference spectra were obtained as an additional method to probe the

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interaction of ligands with the CYP6G3 active site. Chlorfenvinphos, malathion, DDT,

313

endosulfan, and diazinon exhibited type I binding spectra with absorbance maxima near 390

314

nm and minima near 420 nm (Figure 4). Ketoconazole binding exhibited a type II spectrum

315

with a spectral dissociation constant (KS) of 4.3 ± 0.3 µM (Table 2). Endosulfan induced the

316

difference spectrum with the largest absolute amplitude (∆Amax), while chlorfenvinphos

317

bound with the largest spectral binding intensity (∆Amax/KS)(Shimada et al., 2013; Shimada et

318

al., 2009). A weak type I binding spectrum was elicited by imidacloprid, but the Ks value

319

could not be determined accurately (data not shown).

EP

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321

3.5. Metabolism of pesticide substrates by CYP6G3

322

Imidacloprid was metabolized to the 5-hydroxy and olefin metabolites at both 25 and 500 µM

323

substrate concentrations (Figure 5). Diazinon was metabolized to four putative metabolites,

324

two of which were identified as diazoxon and hydroxydiazinon. (Figure 6). The other two

325

putative metabolites could not be further characterized due to limiting quantities.

326

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ACCEPTED MANUSCRIPT 327

4. Discussion

328 329

4.1. CYP6G3 expression and solubility

330 The bicistronic expression vector, pCW6g3-Barnes/fCPR, was constructed with L.

332

cuprina Cyp6g3 and housefly fCPR. The expression yield of CYP6G3 was adequate with the

333

standard expression protocol employed without further optimization. This contrasts markedly

334

with the behaviour of the D. melanogaster CYP6G1, which required specific expression

335

conditions to prevent a loss of holoprotein with a concurrent increase in P420 (Cheesman et

336

al., 2013). Although a minor P420 component was also seen in the CYP6G3 spectra, the

337

facile expression of CYP6G3 may indicate an increased stability of this form compared to

338

CYP6G1.

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339

341 342

4.2. Characterization of the activity of the recombinant enzyme towards marker substrates

TE D

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To identify marker substrates for characterizing CYP6G3, thirteen potential substrates were screened. CYP6G3 exhibited pNAOD and 7ECOD activity. No significant activity was

344

observed toward p-nitrophenol hydroxylation, coumarin-7-hydroxylation, 7-

345

benzyloxyresorufin O-dealkylation, or the dealkylation of several P450-glo substrates

346

(luciferin-ME, -CEE, -H, -BE, -PFBE, -ME-EGE, or -H-EGE).

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Many P450 enzymes exhibit increased catalytic rates in the presence of cytochrome b5

348

(Yamazaki et al., 2002). To determine the effect of fb5 on CYP6G3 activity, pNAOD was

349

used as a probe substrate. The kcat of CYP6G3-mediated pNAOD was increased roughly two

350

fold in the presence of fb5, while the KM was not significantly changed (Fig 2), suggesting an

351

effect on overall turnover but not interactions with substrate. Notably, neither the kcat nor the

15

ACCEPTED MANUSCRIPT KM of CYP6G1-mediated pNAOD was affected by the addition of fb5 (Cheesman et al.,

353

2013). Other insect P450s, including CYP6A1 from M. domestica (Guzov et al., 1996;

354

Murataliev et al., 2008), CYP6CM1vQ from Bemisia tabaci (Karunker et al., 2009) and

355

CYP6M2 from Anopheles gambiae (Stevenson et al., 2011), have shown increased rates of

356

catalysis in the presence of fb5.

357 358

4.3. Screening for pesticide interactions with CYP6G3

360

SC

359

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Oxidative metabolism has been implicated in the resistance of L. cuprina to organochlorine, organophosphorus, benzoylurea, pyrethroid, and carbamate pesticides

362

(Hughes, 1982; Hughes and Devonshire, 1982; Kotze, 1993; Kotze, 1995; Kotze and Sales,

363

1994). Identification and characterization of the specific enzymes involved in L. cuprina

364

oxidative metabolism may be useful to predict and respond to future instances of insecticide

365

resistance in L. cuprina. In particular, CYP6G3, shares 60% and 72% sequence identity with

366

CYP6G1 from D. melanogaster and CYP6G4 from M. domestica, respectively. These P450s

367

have both been implicated in the resistance to multiple pesticides (Feyereisen, 2012; Gao et

368

al., 2012; Hojland et al., 2014). The high degree of sequence identity and the data presented

369

here indicate that the Cyp6G3 gene could confer resistance to multiple insecticides if it were

370

expressed at suitably high levels. The P450 genes of L. cuprina have not been fully annotated

371

as yet (Anstead et al., 2015), so it is not clear whether other P450s in this species might also

372

have this potential. For CYP6G3, two complementary approaches were used to investigate

373

pesticide interaction with the active site, namely inhibition of a marker activity and ligand-

374

induced binding spectra.

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375 376

16

ACCEPTED MANUSCRIPT 377

Since 7ECOD activity was the more sensitive of the two marker substrates metabolized by CYP6G3, this assay was used to screen for inhibition of CYP6G3 activity in high

379

throughput fashion. Activity was supported by CuOOH to avoid interference by NADPH in

380

the high throughput fluorescence assay and allow continuous monitoring of rates. The

381

organochlorine insecticides, DDT and endosulfan, both inhibited CYP6G3-mediated 7ECOD

382

activity and induced type I spectral binding spectra. DDT was used to control flystrike in

383

Australia from 1948 to 1954, after which dieldrin, a related organochlorine compound and

384

more effective insecticide, was preferred (Levot, 1995a). L. cuprina resistance to dieldrin

385

appeared in 1958 (Levot, 1995a). However, the principal, observed genotype of resistance,

386

Rdl, is associated with mutation of a pesticide target, not with oxidative metabolism. Resistant

387

flies have a mutated neuronal GABA receptor, which reduces dieldrin toxicity (McKenzie and

388

Batterham, 1998; Smyth et al., 1992). Nonetheless, the epoxidation of aldrin, another

389

organochlorine insecticide, is a well-characterized reaction catalyzed by L. cuprina

390

microsomes (Kotze, 1993; Kotze, 1995). Furthermore, CYP6G1 has been associated with D.

391

melanogaster resistance to DDT (Daborn et al., 2002) and has been shown to bind both DDT

392

and endosulfan (Cheesman et al., 2013). The current data demonstrate an interaction of DDT

393

and endosulfan with the active site of CYP6G3 and suggest that CYP6G3 could be an

394

additional mechanism of L. cuprina resistance to organochlorine insecticides.

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The organophosphorus insecticides malathion and chlorfenvinphos also inhibited

396

CYP6G3-mediated 7ECOD activity and induced type I binding spectra. Organophosphorus

397

insecticides were used heavily in Australia after resistance developed to organochlorine

398

insecticides. In 1965, about nine years after introduction, resistance to organophosphorus

399

insecticides was observed in Australian populations of L. cuprina (Levot, 1995a). The

400

primary mode of resistance, which has been observed in field samples and in laboratory-

401

evolved resistant lines, involves carboxylesterase-mediated degradation (Claudianos et al.,

17

ACCEPTED MANUSCRIPT 1999; McKenzie and Batterham, 1998). However, several reports have implicated P450

403

enzymes in the ability of L. cuprina to degrade organophosphorus compounds (Hughes and

404

Devonshire, 1982; Kotze, 1995). The inhibition of CYP6G3 activity by chlorfenvinphos and

405

malathion and their tight binding in a type I format are both consistent with these molecules

406

being CYP6G3 substrates. Thus, CYP6G3 is a potential candidate for oxidative degradation

407

of organophosphorus insecticides in L. cuprina.

RI PT

402

Little significant inhibition was observed with lufenuron, carbaryl, dicyclanil, lauric

409

acid, and biphenyl. Similarly to the current work with CYP6G3, at concentrations up to 100

410

µM, biphenyl induced little inhibition with CYP6G1 (Cheesman et al., 2013). Lauric acid is a

411

substrate of D. melanogaster CYP6A8 (Helvig et al., 2004), but does not seem to interact

412

with either the active site of CYP6G3 or CYP6G1 at concentrations up to 100 µM.

413

Lufenuron, a benzoylurea compound, and carbaryl, a carbamate, did not significantly inhibit

414

CYP6G3; however, a slight trend of increasing inhibition is apparent at higher carbaryl and

415

lufenuron concentrations. Finally, dicyclanil, a triazine insecticide, did not inhibit CYP6G3 at

416

concentrations up to 100 uM. Dicyclanil and cyromazine are both currently used to control

417

blowfly in Australia, and the first documented case of cyromazine resistance was recorded

418

recently (Levot, 2012).

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Although imidacloprid is a substrate of CYP6G1 (Fusetto et al., 2017; Hoi et al., 2014; Jouβen et al., 2010), in this work, only weak inhibition of CYP6G3 was seen at millimolar

421

concentrations. Likewise, imidacloprid elicited only a very weak type I binding spectrum.

422

However metabolic experiments confirmed that CYP6G3 metabolized imidacloprid to the 5-

423

hydroxy and olefin metabolites, both of which are toxic in other insects (Fusetto et al., 2017;

424

Nauen et al., 2001). Activity at 500 µM was markedly higher than at 25 µM suggesting

425

CYP6G3 has a low affinity for imidacloprid, consistent with the failure to see significant

426

inhibition of 7ECOD activity.

AC C

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ACCEPTED MANUSCRIPT Diazinon showed a weak Type I binding spectrum with CYP6G3 with a Kd value of

428

23 +/- 3 µM but only inhibited 7ECOD activity at millimolar concentrations, suggesting that

429

it may bind only very weakly to the active site. However CYP6G3 metabolized diazinon to

430

four putative metabolites, two of which were identified as diazoxon and hydroxydiazinon.

431

The apparent metabolites at ~ 6 and 26 min could not be conclusively identified, but it is

432

possible that one may be IMP. The limited inhibition of CYP6G3 by diazinon, its micromolar

433

Kd value and relatively weak type I spectrum is consistent with diazinon binding at the active

434

site in multiple conformations, leading to multiple metabolites.

SC

435

RI PT

427

A role has been proposed for P450s in diazinon metabolism of the phosphorothioate to the toxic oxon metabolite, diazoxon, which is metabolized further by an esterase in resistant

437

L. cuprina populations (Hartley et al., 2006; Newcomb et al., 1997). While CYP6A, CYP6B

438

and CYP12A enzymes have been shown to metabolize diazinon in other species (reviewed in

439

(Feyereisen, 2012)), the diazinon desulfurylase in L. cuprina has not previously been

440

reported. The general P450 inhibitor, piperonyl butoxide, exerted a biphasic effect on

441

diazinon toxicity dependent on the concentration, which may be related to the balance

442

between diazinon bioactivation to the oxon and clearance to the hydroxylated diazinon

443

metabolites (Li et al., 2007a; Mutch and Williams, 2006; Wilson et al., 1999). The data

444

presented here suggest that CYP6G3 is involved in both activation of diazinon to diazoxon

445

and its detoxification to hydroxydiazinon metabolites consistent with this biphasic behaviour.

447

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446

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4.4. Concluding comments

448 449

The in vitro characterization of P450 enzymes can provide valuable information about the

450

degradative capabilities of industrially-relevant pests and offer clues to the design of more

451

effective insecticides. The heterologous expression of CYP6G3, a P450 enzyme from the

19

ACCEPTED MANUSCRIPT economically important pest L. cuprina, enabled the characterization CYP6G3 activity

453

towards pesticides commonly used for flystrike, and revealed that CYP6G3 can metabolize

454

imidacloprid and diazinon. The inhibition of marker activity and type I ligand binding spectra

455

shown by the organochlorine insecticides, DDT and endosulfan, and by the

456

organophosphorous insecticides, chlorfenvinphos and malathion, are strong indicators that

457

CYP6G3 is also capable of degrading these molecules. However, a conclusive demonstration

458

that these molecules are indeed substrates of CYP6G3 will require additional work to identify

459

reaction products. This work is a step toward a complete characterization of the catalytic

460

capabilities of CYP6G3 and other xenobiotic-metabolizing P450 enzymes from L. cuprina.

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ACCEPTED MANUSCRIPT Acknowledgments

463

Matthew Traylor’s contribution was supported by a Fulbright Scholarship, funded by the

464

Australian-American Fulbright Commission. Roberto Fusetto received scholarship support

465

from the University of Melbourne. Financial support from the University of Melbourne

466

Interdisciplinary Seed Grant program is gratefully acknowledged. We thank Dr. Phillip

467

Daborn for his assistance in providing the cDNA for CYP6G3.

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ACCEPTED MANUSCRIPT 469 470

Figure legends

471 Figure 1. CO-difference spectrum of microsomes containing CYP6G3.

473

RI PT

472

Figure 2. A) Initial rate of 7-ethoxycoumarin O-dealkylation (7ECOD) by CYP6G3 supported

475

by cumene hydroperoxide. B) Initial rate of p-nitroanisole O-dealkylation (pNAOD) by

476

CYP6G3 supported by fCPR (●) with and (○) without fb5.

SC

474

477

Figure 3. Inhibition of CYP6G3-mediated 7ECOD (1 mM 7EC) by varying concentrations of

479

A) chlorfenvinphos, B) malathion, C) endosulfan, D), DDT, and E) ketoconazole.

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480

Figure 4. Ligand-induced difference spectra and best fit to the concentration dependence of

482

the difference magnitude for CYP6G3 binding. A) and B) chlorfenvinphos, C) and D)

483

malathion, E) and F) ketoconazole, G) and H) DDT, I) and J) endosulfan, and K) and L)

484

diazinon.

EP

485

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481

Figure 5. Imidacloprid metabolism by CYP6G3. Bacterial membranes containing

487

recombinant CYP6G3 (0.10 µM P450) and fCPR (0.18 µM) were incubated with either 25

488

µM or 500 µM imidacloprid for two hours under conditions supporting P450 catalysis.

489

Controls lacked P450 (CPR only) or an NADPH-generating system (-NGS), were quenched at

490

time zero, or contained only substrate and buffer (substrate only). Data represent the means

491

+/- S.D. of three independent experiments. Metabolite formation was significantly higher in

492

complete reactions compared to the highest control (CPR only) at the p < 0.05 level for assays

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ACCEPTED MANUSCRIPT 493

done with 25 µM substrate concentration for both metabolites and at the p < 0.01 (5-

494

hydroxyimidacloprid) and p < 0.001 (imidacloprid olefin) levels for 500 µM imidacloprid.

495 Figure 6. HPLC Chromatogram from extracts of reactions exploring diazinon metabolism by

497

CYP6G3. Bacterial membranes containing recombinant CYP6G3 (0.50 µM P450) and fCPR

498

(0.75 µM) were incubated with 100 µM diazinon for two hours under conditions supporting

499

P450 catalysis (upper, blue trace). Controls lacked P450 (fCPR only; lower, pink trace) or an

500

NADPH-generating system (-NGS, middle, black trace).

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501

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ACCEPTED MANUSCRIPT 503

References Andersen, J.F., Utermohlen, J.G., Feyereisen, R., 1994. Expression of house fly CYP6A1 and

505

NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-

506

metabolizing P450 system. Biochemistry 33, 2171-2177.

507

Anstead, C.A., Korhonen, P.K., Young, N.D., Hall, R.S., Jex, A.R., Murali, S.C., Hughes,

508

D.S.T., Lee, S.F., Perry, T., Stroehlein, A.J., Ansell, B.R.E., Hofmann, A., Qu, J., Dugan, S.,

509

Lee, S.L., Chao, H., Dinh, H., Han, Y., Doddapaneni, H.V., Worley, K.C., Muzyny, D.M.,

510

Ionnidis, P., Waterhouse, R.M., Zdobnov, E.M., James, P.J., Bagnall, N.H., Kotze, A.C.,

511

Gibbs, R.A., Richards, S., Batterham, P. , Gasser, R.B., 2015. Lucilia cuprina genome unlocks

512

parasitic fly biology to underpin future interventions. Nat. Commun. 6, 7344.

513

Barnes, H.J., Arlotto, M.P., Waterman, M.R., 1991. Expression and enzymatic activity of

514

recombinant cytochrome P450 17a-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci.

515

USA 88, 5597-5601.

516

Bauer, S., Shiloach, J., 1974. Maximal exponential growth rate and yield of E.coli obtainable

517

in a bench-scale fermentor. Biotechnol. Bioeng. 16, 933-941.

518

Cali, J.J., Ma, D., Sobol, M., Simpson, D.J., Frackman, S., Good, T.D., Daily, W.J., Liu, D.,

519

2006. Luminogenic cytochrome P450 assays. Expert Opin. Drug Metab. Toxicol. 2, 629-645.

520

Capinera, J., 2008. Australian Sheep Blowfly, Lucilia cuprina Wiedemann (Diptera:

521

Calliphoridae), in: Capinera, J. (Ed.), Encyclopedia of Entomology. Springer, Netherlands,

522

pp. 335-338.

523

Chang, T.K., Crespi, C.L., Waxman, D.J., 2006. Spectrophotometric analysis of human

524

CYP2E1-catalyzed p-nitrophenol hydroxylation, in: Phillips, I.R., Shephard, E.A. (Eds.),

525

Cytochrome P450 Protocols. Humana Press, Totowa, N.J., pp. 127-131.

526

Cheesman, M.J., Traylor, M.J., Hilton, M.E., Richards, K.E., Taylor, M.C., Daborn, P.J.,

527

Russell, R.J., Gillam, E.M.J., Oakeshott, J.G., 2013. Soluble and membrane-bound

AC C

EP

TE D

M AN U

SC

RI PT

504

24

ACCEPTED MANUSCRIPT Drosophila melanogaster CYP6G1 expressed in E. coli: purification, activity, and binding

529

properties toward multiple pesticides. Insect Biochem. Molec. Biol. 43, 455–465.

530

Claudianos, C., Russell, R.J., Oakeshott, J.G., 1999. The same amino acid substitution in

531

orthologous esterases confers organophosphate resistance on the house fly and a blowfly.

532

Insect Biochem. Molec. Biol. 29, 675-686.

533

Daborn, P.J., Lumb, C., Boey, A., Wong, W., Ffrench-Constant, R.H., Batterham, P., 2007.

534

Evaluating the insecticide resistance potential of eight Drosophila melanogaster cytochrome

535

P450 genes by transgenic over-expression. Insect Biochem. Molec. Biol. 37, 512-519.

536

Daborn, P.J., Yen, J.L., Bogwitz, M.R., Le Goff, G., Feil, E., Jeffers, S., Tijet, N., Perry, T.,

537

Heckel, D., Batterham, P., Feyereisen, R., Wilson, T.G., ffrench-Constant, R.H., 2002. A

538

single P450 allele associated with insecticide resistance in Drosophila. Science 297, 2253-

539

2256.

540

Ellison, C.A., Tian, Y., Knaak, J.B., Kostyniak, P.J., Olson, J.R., 2012. Human hepatic

541

cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion

542

and diazinon. Drug Metab. Dispos. 40, 1-5.

543

Estabrook, R.W., Werringloer, J., 1978. The measurement of difference spectra: Application

544

to the cytochromes of microsomes. Methods Enzymol. 52, 212-220.

545

Feyereisen, R., 2012. Insect CYP genes and P450 enzymes, in: Gilbert, L.I. (Ed.), Insect

546

Molecular Biology and Biochemistry. Elsevier B.V., London.

547

Fusetto, R., Denecke, S., Perry, T., O’Hair, R.A.J., Batterham, P., 2017. Partitioning the roles

548

of CYP6G1 and gut microbiota in the metabolism of the insecticide imidacloprid in

549

Drosophila melanogaster. Sci Rep in press.

550

Gao, Q., Li, M., Sheng, C.F., Scott, J.G., Qiu, X.H., 2012. Multiple cytochrome P450s

551

overexpressed in pyrethroid resistant house flies (Musca domestica). Pestic. Biochem.

552

Physiol. 104, 252-260.

AC C

EP

TE D

M AN U

SC

RI PT

528

25

ACCEPTED MANUSCRIPT Gillam, E.M.J., Hunter, D.J.B., 2007. Chemical Defence and Exploitation: Biotransformation

554

of Xenobiotics by Cytochrome P450 Enzymes, in: Sigel, A., Sigel, H., Sigel, R.K.O. (Eds.),

555

Metal Ions in Life Sciences: The Ubiquitous Roles of Cytochrome P450 Proteins. John Wiley

556

& Sons, pp. 477-560.

557

Guengerich, F.P., 2001. Common and uncommon cytochrome P450 reactions related to

558

metabolism and chemical toxicity. Chem. Res. Toxicol. 14, 611-650.

559

Guzov, V.M., Houston, H.L., Murataliev, M.B., Walker, F.A., Feyereisen, R., 1996.

560

Molecular cloning, overexpression in Escherichia coli, structural and functional

561

characterization of house fly cytochrome b5. J. Biol. Chem. 271, 26637-26645.

562

Hansen, L.G., Hodgson, E., 1971. Biochemical characteristics of insect microsomes - N-

563

demethylation and O-demethylation. Biochem. Pharmacol. 20, 1569-1578.

564

Hartley, C.J., Newcomb, R.D., Russell, R.J., Yong, C.G., Stevens, J.R., Yeates, D.K., La

565

Salle, J., Oakeshott, J.G., 2006. Amplification of DNA from preserved specimens shows

566

blowflies were preadapted for the rapid evolution of insecticide resistance. Proc. Natl. Acad.

567

Sci. USA 103, 8757-8762.

568

Heath, A.C.G., Levot, G.W., 2015. Parasiticide resistance in flies, lice and ticks in New

569

Zealand and Australia: mechanisms, prevalence and prevention. N. Z. Vet. J. 63, 199-210.

570

Heckel, D.G., 2010. Molecular genetics of insecticide resistance in Lepidoptera, in:

571

Goldsmith, M.R., Marec, F. (Eds.), Molecular Biology and Genetics of the Lepidoptera. CRC

572

Press, Boca Raton, pp. 239-270.

573

Helvig, C., Tijet, N., Feyereisen, R., Walker, F.A., Restifo, L.L., 2004. Drosophila

574

melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation.

575

Biochem Biophys Res Commun 325, 1495-1502.

576

Hoi, K.K., Daborn, P.J., Battlay, P., Robin, C., Batterham, P., O'Hair, R.A.J., Donald, W.A.,

577

2014. Dissecting the Insect Metabolic Machinery Using Twin Ion Mass Spectrometry: A

AC C

EP

TE D

M AN U

SC

RI PT

553

26

ACCEPTED MANUSCRIPT Single P450 Enzyme Metabolizing the Insecticide Imidacloprid in Vivo. Anal. Chem. 86,

579

3525-3532.

580

Hojland, D.H., Jensen, K.M.V., Kristensen, A., 2014. A comparative study of P450 gene

581

expression in field and laboratory Musca domestica L. strains. Pest Manag. Sci. 70, 1237-

582

1242.

583

Hughes, P.B., 1982. Organo-phosphorus resistance in the sheep blowfly, Lucilia cuprina

584

(wiedemann) (Diptera, Calliphoridae) - a genetic-study incorporating synergists. Bull.

585

Entomol. Res. 72, 573-582.

586

Hughes, P.B., Devonshire, A.L., 1982. The biochemical basis of resistance to organo-

587

phosphorus insecticides in the sheep blowfly, Lucilia cuprina. Pestic. Biochem. Physiol. 18,

588

289-297.

589

Jefcoate, C.R., 1978. Measurement of substrate and inhibitor binding to microsomal

590

cytochrome P-450 by optical-difference spectroscopy. Methods Enzymol. 52, 258-279.

591

Joshua, E., Turnbull, G., 2015. Chemicals registered to treat lice and flystrike, New South

592

Wales Department of Primary Industries, p. Fact Sheet.

593

Jouβen, N., Schuphan, I., Schmidt, B., 2010. Metabolism of methoxychlor by the P450

594

monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after

595

expression in cell cultures of Nicotiana tabacum. Chem. Biodivers. 7, 722-735.

596

Karunker, I., Morou, E., Nikou, D., Nauen, R., Sertchook, R., Stevenson, B.J., Paine, M.J.I.,

597

Morin, S., Vontas, J., 2009. Structural model and functional characterization of the Bemisia

598

tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid

599

resistance. Insect Biochem. Molec. Biol. 39, 697-706.

600

Kita, K., Konishi, K., Anraku, Y., 1984. Terminal oxidases of escherichia-coli aerobic

601

respiratory-chain .2. Purification and properties of cytochrome b558-d complex from cells

AC C

EP

TE D

M AN U

SC

RI PT

578

27

ACCEPTED MANUSCRIPT grown with limited oxygen and evidence of branched electron-carrying systems. J. Biol.

603

Chem. 259, 3375-3381.

604

Kotze, A.C., 1993. Cytochrome P450 monooxygenases in larvae of insecticide-susceptible

605

and -resistant strains of the Australian sheep blowfly, Lucilia cuprina. Pestic. Biochem.

606

Physiol. 46, 65-72.

607

Kotze, A.C., 1995. Induced insecticide tolerance in larvae of Lucilia cuprina (wiedemann)

608

(Diptera, Calliphoridae) following dietary phenobarbital treatment. J. Aust. Entomol. Soc.

609

34, 205-209.

610

Kotze, A.C., Sales, N., 1994. Cross-resistance spectra and effects of synergists in insecticide-

611

resistant strains of Lucilia cuprina (Diptera, Calliphoridae). Bull. Entomol. Res. 84, 355-360.

612

Kotze, A.C., Sales, N., Barchia, I.M., 1997. Diflubenzuron tolerance associated with

613

monooxygenase activity in field strain larvae of the Australian sheep blowfly

614

(Diptera:Calliphoridae). J Econ Entomol 90, 15-20.

615

Kouloumbos, V.N., Tsipi, D.F., Hiskia, A.E., Nikolic, D., van Breemen, R.B., 2003.

616

Identification of photocatalytic degradation products of diazinon in TiO2 aqueous suspensions

617

using GC/MS/MS and LC/MS with quadrupole time-of-flight mass spectrometry. J Am Soc

618

Mass Spectrom 14, 803-817.

619

Lee, S.F., Chen, Z., McGrath, A., Good, R.T., Batterham, P., 2011. Identification, analysis,

620

and linkage mapping of expressed sequence tags from the Australian sheep blowfly. BMC

621

Genomics 12, 406.

622

Levot, G.W., 1995a. Resistance and the control of sheep ectoparasites. Int J Parasitol 25,

623

1355-1362.

624

Levot, G.W., 1995b. Resistance and the control of sheep ectoparasites. Int. J. Parasit. 25,

625

1355-1362.

AC C

EP

TE D

M AN U

SC

RI PT

602

28

ACCEPTED MANUSCRIPT Levot, G.W., 2012. Cyromazine resistance detected in Australian sheep blowfly. Aust. Vet. J.

627

90, 433-437.

628

Li, A.Y., Guerrero, F.D., Pruett, J.H., 2007a. Involvement of esterases in diazinon resistance

629

and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans

630

(Diptera : Muscidae). Pestic. Biochem. Physiol. 87, 147-155.

631

Li, X.C., Schuler, M.A., Berenbaum, M.R., 2007b. Molecular mechanisms of metabolic

632

resistance to synthetic and natural xenobiotics. Annual Review of Entomology 52, 231-253.

633

McKenzie, J.A., Batterham, P., 1998. Predicting insecticide resistance: mutagenesis, selection

634

and response. Philos. Trans. R. Soc. Lond. Ser. B-Biol. Sci. 353, 1729-1734.

635

Murataliev, M.B., Arino, A., Guzov, V.M., Feyereisen, R., 1999. Kinetic mechanism of

636

cytochrome P450 reductase from the house fly (Musca domestica). Insect Biochem. Molec.

637

Biol. 29, 233-242.

638

Murataliev, M.B., Guzov, V.M., Walker, F.A., Feyereisen, R., 2008. P450 reductase and

639

cytochrome b(5) interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis.

640

Insect Biochem. Molec. Biol. 38, 1008-1015.

641

Mutch, E., Williams, F.M., 2006. Diazinon, chlorpyrifos and parathion are metabolised by

642

multiple cytochromes P450 in human liver. Toxicology 224, 22-32.

643

Nauen, R., Ebbinghaus-Kintscher, U., Schmuck, R., 2001. Toxicity and nicotinic

644

acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera

645

(Hymenoptera: Apidae). . Pest Management Scies 57, 577-586.

646

Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997.

647

A single amino acid substitution converts a carboxylesterase to an organophosphorus

648

hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94,

649

7464-7468.

AC C

EP

TE D

M AN U

SC

RI PT

626

29

ACCEPTED MANUSCRIPT Nishihara, K., Kanemori, M., Kitagawa, M., Yanagi, H., Yura, T., 1998. Chaperone

651

coexpression plasmids: Differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-

652

GroES in assisting folding of an allergen of Japanese Cedar pollen, Cryj2, in Escherichia coli.

653

Appl. Environ. Microbiol. 64, 1694-1699.

654

Notley, L.M., de Wolf, C.J.F., Wunsch, R.M., Lancaster, R.G., Gillam, E.M.J., 2002.

655

Bioactivation of tamoxifen by recombinant human cytochrome P450 enzymes. Chem. Res.

656

Toxicol. 15, 614-622.

657

Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver microsomes. I.

658

Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370-2378.

659

Phillips, C.J.C., 2009. A review of mulesing and other methods to control flystrike (cutaneous

660

myiasis) in sheep. Anim. Welf. 18, 113-121.

661

PisaniBorg, E., Cuany, A., Brun, A., Amichot, M., Fournier, D., Berge, J.B., 1996. Oxidative

662

degradation of diazinon by Drosophila: Metabolic changes associated with insecticide

663

resistance and induction. Pestic. Biochem. Physiol. 54, 56-64.

664

Shimada, T., Kim, D., Murayama, N., Tanaka, K., Takenaka, S., Nagy, L.D., Folkmann,

665

L.M., Foroozesh, M., Komori, M., Yamazaki, H., Guengerich, F.P., 2013. Binding of diverse

666

environmental chemicals with human cytochromes P450 2A13, 2A6, and 1B1 and enzyme

667

inhibition. Chem. Res. Toxicol. 26, 517–528.

668

Shimada, T., Tanaka, K., Takenaka, S., Foroozesh, M.K., Murayama, N., Yamazaki, H.,

669

Guengerich, F.P., Komori, M., 2009. Reverse type I binding spectra of human cytochrome

670

P450 1B1 induced by flavonoid, stilbene, pyrene, naphthalene, phenanthrene, and biphenyl

671

derivatives that inhibit catalytic activity: A structure-function relationship study. Chem. Res.

672

Toxicol. 22, 1325–1333.

673

Shishido, T., Usui, K., Fukami, J., 1972. Oxidative metabolism of diazinon by microsomes

674

from rat liver and cockroach fat body. Pestic. Biochem. Physiol. 2, 27-&.

AC C

EP

TE D

M AN U

SC

RI PT

650

30

ACCEPTED MANUSCRIPT Smyth, K.A., Parker, A.G., Yen, J.L., McKenzie, J.A., 1992. Selection of dieldrin-resistant

676

strains of Lucilia cuprina (Diptera, Calliphoridae) after ethyl methanesulfonate mutagenesis

677

of a susceptible strain. J. Econ. Entomol. 85, 352-358.

678

Stevenson, B.J., Bibby, J., Pignatelli, P., Muangnoicharoen, S., O'Neill, P.M., Lian, L.Y.,

679

Muller, P., Nikou, D., Steven, A., Hemingway, J., Sutcliffe, M.J., Paine, M.J.I., 2011.

680

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids:

681

Sequential metabolism of deltamethrin revealed. Insect Biochem. Molec. Biol. 41, 492-502.

682

Stresser, D.M., Turner, S.D., Blanchard, A.P., Miller, V.P., Crespi, C.L., 2002. Cytochrome

683

P450 fluorometric substrates: Identification of isoform-selective probes for rat CYP2D2 and

684

human CYP3A4. Drug Metab. Dispos. 30, 845-852.

685

Wall, R., 2012. Ovine cutaneous myiasis: Effects on production and control. Vet. Parasitol.

686

189, 44-51.

687

Waxman, D.J., Chang, T.K.H., 2006a. Spectrofluorometric analysis of CYP2A6-catalyzed

688

coumarin 7-hydroxylation in: Phillips, I.R., Shephard, E.A. (Eds.), Cytochrome P450

689

Protocols. Humana Press, Totowa N.J., pp. 91-96.

690

Waxman, D.J., Chang, T.K.H., 2006b. Use of 7-ethoxycoumarin to monitor multiple enzymes

691

in the human CYP1, CYP2, and CYP3 families, in: Phillips, I.R., Shephard, E.A. (Eds.),

692

Cytochrome P450 Protocols. Humana Press, Totowa N.J., pp. 153-156.

693

Wilson, J.A., Clark, A.G., Haack, N.A., 1999. Effect of piperonyl butoxide on diazinon

694

resistance in field strains of the sheep blowfly, Lucilia cuprina (Diptera : Calliphoridae), in

695

New Zealand. Bull. Entomol. Res. 89, 295-301.

696

Yamazaki, H., Nakamura, M., Komatsu, T., Ohyama, K., Hatanaka, N., Asahi, S., Shimada,

697

N., Guengerich, F.P., Shimada, T., Nakajima, M., Yokoi, T., 2002. Roles of NADPH-P450

698

reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12

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ACCEPTED MANUSCRIPT 699

recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein

700

Express. Purif. 24, 329-337.

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32

ACCEPTED MANUSCRIPT Tables

Table 1 Steady-state kinetic constants of 6G3 7ECOD activity supported by CuOOH and of

RI PT

6G3 pNAOD activity supported by fCPR in the presence and absence of fb5 Activity

kcata

6G3 CuOOH

7ECOD

1.7 ± 0.1

1042 ± 96

6G3 fCPR NADPH

pNAOD

9.6 ± 0.2

9.8 ± 1.1

6G3 fCPR fb5

pNAOD

17.8 ± 0.5

13.4 ± 1.9

nmol / min / nmol P450

b

µM

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a

KMb

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NADPH

SC

System

33

ACCEPTED MANUSCRIPT Table 2 IC50 values for the inhibition of CYP6G3 7ECOD activity and spectral binding parameters for ligand binding to CYP6G3 KS b

Hill

∆Amax

Coefficient endosulfan

0.23

1.4 +/- 0.1

n.a.c

∆Amax / KS

RI PT

IC50a

Ligand

0.064 +/-

0.046

0.001

5.5

4.3 +/- 0.3

1.6 +/- 0.1

0.019 +/-

SC

ketoconazole

0.0044

0.001

26.8

malathion

>100

5.4 +/- 0.4

28 +/- 1

TE D

DDT

>100

1.0 +/- 0.1

n.a.

M AN U

chlorfenvinphos

n.a.

n.a.

0.056 +/-

0.056

0.001 0.030 +/-

0.0056

0.001 0.046 +/-

0.0016

0.001

n.d.d

n.a.

n.d.

n.d.

>100

n.d.

n.a.

n.d.

n.d.

>100

n.d.

n.a.

n.d.

n.d.

imidacloprid

>100

n.d.

n.a.

n.d.

n.d.

biphenyl

>100

n.d.

n.a.

n.d.

n.d.

diazinon

>100

23 +/- 3

n.a.

0.0167

0.0007

carbaryl

AC C

dicyclanil

EP

>100

lufenuron

+/0.0006 a

µM, IC50 values were determined using 1 mM 7ECOD supported by 500 µM

CuOOH

34

ACCEPTED MANUSCRIPT b

µM

c

not applicable; data were best fit using a simple rectangular hyperbolic binding

curve

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not determined

AC C

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35

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT The cytochrome P450, CYP6G3, has been characterised from the Australian Sheep Blowfly, an important agricultural pest. Recombinant CYP6G3 is catalytically active towards marker substrates and stimulated by cytochrome b5.

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CYP6G3 binds and is inhibited by the insecticides DDT, endosulfan, malathion and chlorfenvinphos.

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CYP6G3 metabolizes imidacloprid to the 5-hydroxy- and olefin metabolites and diazinon to diazoxon and hydroxydiazinon.