Accepted Manuscript

Recombinant expression and characterization of 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: 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 CYP6G3: Activity and binding properties toward multiple pesticides, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.09.004.

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ACCEPTED ACCEPTED MANUSCRIPT 1 Recombinant expression and characterization of Lucilia cuprina CYP6G3:

2 activity and binding properties toward multiple pesticides

3

4 Matthew J. Traylor a, Jong-Min Baek a, Katelyn E. Richards a, Roberto Fusetto b, W. Huang,

5 Peter Josh a, Zhengzhong Chen b, Padma Bollapragada a, Richard A. J. O’Hair b, Philip

6 Batterham b, Elizabeth M.J. Gillam a*

7

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

9 Australia

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

11

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

13 [email protected] MANUSCRIPT

ACCEPTED

1 ACCEPTED MANUSCRIPT 14 Abstract

15

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

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

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

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

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

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

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

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

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

25 addition of house fly cytochrome b5 enhanced the k cat for p-nitroanisole dealkylation

-1 26 approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min ) with little effect on K M (13 ± 1 vs 10 ± 27 1 µM). Inhibition studies and difference spectrosco MANUSCRIPTpy revealed that the organochlorine 28 compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and

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

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

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

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

33 assays, with a Kd value of 23 +/- 3 µM. CYP6G3 metabolised diazinon to the diazoxon and 34 hydroxydiazinonACCEPTED metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, 35 consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and

36 neonicotinoid insecticides in other .

37

2 ACCEPTED MANUSCRIPT 38 Keywords: cytochrome P450, CYP6G3, Lucilia cuprina , Australian Sheep Blowfly,

39 insecticide resistance, diazinon, imidacloprid

40

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

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

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

44 reductase from Musca domestica ; fb 5 – cytochrome b5 from Musca domestica ; IPTG -

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

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

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

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

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

50 ester; luciferin-PFBE - luciferin 6’-pentafluorobenzyl ether; P450 – cytochrome P450; PMSF 51 - phenylmethylsulfonyl fluoride; pNAOD – p-nitroanisole MANUSCRIPT O-dealkylation; pNP – p- 52 nitrophenol; pNA – p-nitroanisole.

53

ACCEPTED

3 ACCEPTED MANUSCRIPT 54 1. Introduction

55

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

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

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

59 2012; Phillips, 2009). Lucilia cuprina , the Australian sheep blowfly, causes 90% of these

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

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

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

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

64 Traditionally, the process of mulesing, which involves surgically removing skin folds

65 from areas of the sheep where the most commonly oviposit, is used as an effective

66 method to prevent strike infections. However, mulesing has raised ethical concerns and is 67 now illegal in many countries (Phillips, 2009). Che MANUSCRIPTmical methods of flystrike treatment have 68 since been applied, with organochlorine, organophosphorus, and triazine pesticides all having

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

70 organochlorine, organophosphorus, benzoylurea, pyrethroid, and carbamate pesticides

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

72 1994), further complicating the management of flystrike in the grazing industry (Heath and

73 Levot, 2015; Levot, 1995b, a; Phillips, 2009). 74 CytochromeACCEPTED P450 enzymes are a ubiquitous class of hemoproteins present in all 75 classes of life. These enzymes catalyse over 60 types of reactions and are responsible for a

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

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

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

4 ACCEPTED MANUSCRIPT 79 generally presumed and sometimes proven that they confer resistance by detoxifying

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

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

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

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

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

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

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

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

88 diazoxon or hydroxydiazinon are yet to be identified.

89 Pesticide resistance in the model fly species Drosophila melanogaster has been

90 extensively characterized (Feyereisen, 2012). In Drosophila , CYP6G1 has been associated

91 with resistance to many other classes of insecticides (Cheesman et al., 2013; Daborn et al., 92 2007; Feyereisen, 2012) including the neonicotinoid MANUSCRIPT, imidacloprid (Fusetto et al., 2017; Hoi et 93 al., 2014). The proposed CYP6G1 ortholog in houseflies, CYP6G4 has similarly been

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

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

96 P450s have been implicated in L. cuprina resistance for decades (Hughes, 1982;

97 Hughes and Devonshire, 1982; Kotze and Sales, 1994; Kotze et al., 1997) but no biochemical

98 studies of specific L. cuprina P450 enzymes have been done to date. Since CYP6G3 from L. 99 cuprina is in theACCEPTED same subfamily as CYP6G1 and CYP6G4 we hypothesized that it may also 100 play a role in insecticide metabolism. The objective of this work was to develop a bacterial

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

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

5 ACCEPTED MANUSCRIPT 103 the xenobiotic detoxification capacities of L. cuprina . In particular, we sought to determine

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

105

106 2. Materials and Methods

107

108 2.1. Materials

109

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

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

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

113 (Madison, WI), and DH5 α F′IQ ™ Max Efficiency cells were purchased from Invitrogen

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

115 and QIAquick PCR Purification and Gel Extraction Kits were from Qiagen (Valencia, CA). 116 P450-glo reagents were from Promega (Madison, MANUSCRIPTWI). The pesticides chlorfenvinphos, 117 endosulfan and dicyclanil were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,

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

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

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

13 121 obtained from AnalaR (supplied by BDH, Poole, Great Britain). C6-imidacloprid was

122 provided by IsoSciences (PA, USA). 5-hydroxy-imidacloprid and olefin-imidacloprid were 123 purchased from ACCEPTEDBayer AG (Leverkusen, North Rhine-Westphalia, Germany). HPLC grade 124 acetonitrile and HPLC grade methanol were obtained from Merck (Kenilworth, NJ, U.S.A.).

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

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

6 ACCEPTED MANUSCRIPT 127 chemicals and reagents were of the highest quality commercially available from local

128 suppliers and were used as purchased.

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

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

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

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

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

134

135 2.2. Construction of the pCW6G3-Barnes/fCPR expression plasmid

136

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

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

139 vector. A bacterial expression plasmid containing the cDNAs for CYP6G3 and housefly 140 cytochrome P450 reductase (fCPR) arranged in a biciMANUSCRIPTstronic format was constructed using the 141 pCW6G1-Barnes/fCPR vector (Cheesman et al., 2013) from which the 6G1 cDNA had been

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

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

144 GGAGGTGGACATATGGCTCTGTTATTAGCAGTTTTTTTCATTACCTGTATTCTTGG-

145 3’) that incorporated an NdeI restriction site into the start codon and encoded the

146 MALLLAVFL N-terminal sequence to facilitate expression (Barnes et al., 1991). The reverse 147 primer (5’- ACCEPTED 148 GTGGTGGTGGTCGACTTAACAAGTATATTTTTATCATAGAGATTATCTCTTATCAC

149 -3’) added a SalI restriction site to the end of the open reading frame to enable subcloning into

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

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

7 ACCEPTED MANUSCRIPT 152 hexa-histidine affinity tag connected with a serine/threonine linker to facilitate future

153 purification.

154

155 2.3. Expression in E. coli and preparation of bacterial membranes

156

157 The pCW6G3-Barnes/fCPR plasmid was co-expressed in DH5 αF’IQ TM E. coli cells

158 containing the chaperone expression plasmid pGro7 (Nishihara et al., 1998) using a standard

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

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

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

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

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

164 shaking for 5 h before induction with the addition of 4 mg/ml arabinose, 1 mM IPTG, and 0.5 165 mM δ-ALA. Expression cultures were incubated withMANUSCRIPT shaking at 25°C for a further 43 h (48 h 166 total) before harvest. Bacterial membranes containing recombinant CYP6G3 and fCPR were

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

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

169 To supplement enzymatic reactions with controlled and reproducible amounts of P450

170 reductase, membranes containing fCPR were generated from bacteria expressing recombinant

171 fCPR alone from the pCW/fCPR vector (Cheesman et al., 2013) in the same manner as 172 described above.ACCEPTED Additionally, membranes containing housefly cytochrome b5 (fb 5) were 173 generated similarly from cultures expressing the pCb5 vector (Cheesman et al., 2013; Guzov

174 et al., 1996).

175 The concentration of CYP6G3 was measured in membrane preparations via Fe (II) .CO

176 vs Fe(II) difference spectroscopy (Omura and Sato, 1964). Microsomal fCPR content was

8 ACCEPTED MANUSCRIPT 177 measured using cyt c as a surrogate electron acceptor and assuming a specific activity of 3024

-1 -1 178 nmol cyt c reduced min nmol fCPR (Murataliev et al., 1999). Microsomal fb 5 content was

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

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

181

182 2.4. Characterization of activity toward marker substrates

183

184 Thirteen potential marker substrates were screened for CYP6G3 activity. The

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

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

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

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

189 luciferin-ME, -CEE, -H, –BE, -PFBE, -ME-EGE and –H-EGE were assayed as described 190 previously (Cheesman et al., 2013). All reactions iMANUSCRIPTncluded 50 nM CYP6G3 and were initiated 191 by the addition of either 500 µM cumene hydroperoxide (CuOOH) for the fluorogenic

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

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

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

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

196 fluorescence intensity measurements in real time without interference from NADPH 197 fluorescence. TheACCEPTED electron transfer proteins fCPR and fb 5 were supplemented where indicated 198 to a final level of 250 nM and 500 nM, respectively, in reactions supported by the NADPH-

199 regenerating system.

200 Catalytic constants were measured for p-nitroanisole O-dealkylation (pNAOD) in the

201 presence and absence of 500 nM fb 5 as described previously (Cheesman et al., 2013).

9 ACCEPTED MANUSCRIPT 202 Catalytic constants were measured for 7-ethoxycoumarin O-dealkylation (7ECOD) supported

203 by CuOOH in the absence of fb 5 or additional fCPR above the level already present in the

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

205 without additional fCPR or fb 5 in the presence of 100, 10, 1, 0.1, 0.01 and 0 µM inhibitor.

206 GraphPad prism was used to calculate IC 50 values using the following equation,

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

208

209 2.5. Ligand binding experiments

210

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

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

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

214 mM Tris acetate, 250 mM sucrose, 0.25 mM EDTA, pH 7.6) and the membrane suspension 215 was split between sample and reference cuvettes. LiMANUSCRIPTgands were dissolved in methanol and 216 sequential, equal additions of dissolved ligand and methanol were added to the sample and

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

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

219

220 2.6. Characterization of imidacloprid metabolism

221 CYP6G3-mediated imidacloprid metabolism was assessed in vitro by incubating

12 13 222 bacterial membranesACCEPTED with C6 and C6- imidacloprid in a 50:50 ratio to allow the 223 identification of imidacloprid metabolites through the use of the twin-ion method described

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

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

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

10 ACCEPTED MANUSCRIPT 227 with 25 µM or 500 µM imidacloprid (total concentration) in 100 mM potassium phosphate

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

229 (10 mM glucose-6-phosphate, 250 µM NADP +, and 0.5 U/ml glucose-6-phosphate

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

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

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

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

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

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

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

237 16 000 x g and the supernatants were recovered. A second extraction was performed with 500

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

239 residues were stored in the freezer (-20°C) prior to analysis of the metabolites. 240 Imidacloprid metabolites were analysed as described MANUSCRIPT previously by Fusetto et al., (2017). 241 Briefly, the dried extracts were resuspended in HPLC grade water, and analysed using an

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

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

244 6520 Q-TOF mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA).

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

246 a 70:30:0.1% mixture of acetonitrile, H 2O, and formic acid, respectively (Phase 2). 247 Imidacloprid, andACCEPTED its 5-hydroxyimidacloprid (5OH) and imidacloprid olefin (Olefin) 248 metabolites were analysed in both positive and negative mode and quantified using calibration

249 curves (R 2=0.989, R 2=0.999 and R 2=0.999 respectively) generated by multiple injections of

250 the pure standard at different concentrations.

251

11 ACCEPTED MANUSCRIPT 252 2.7. Characterization of diazinon metabolism

253 Incubations were undertaken as described above with 100 µM diazinon, 0.5 µM

254 CYP6G3 and 0.75 µM fCPR. Reactions were stopped after 2 h with two volumes of

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

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

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

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

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

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

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

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

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

264 90% acetonitrile. The separation gradient was 0-11 minutes 2% B; 11-14 minutes 45% B; 14- 265 16 minutes 95% B. Compound identification was MANUSCRIPT confirmed by matching MS2 fragmentation 266 data against the MassBank database (http://www.massbank.jp) and data previously published

267 (Kouloumbos et al., 2003).

268

269

270 3. Results

271 272 3.1. CYP6G3 expression,ACCEPTED membrane preparation, and protein quantitation 273

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

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

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

12 ACCEPTED MANUSCRIPT 277 expression in E. coli . Expression yields from the pCW6G3-Barnes/fCPR vector were

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

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

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

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

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

283 cytochromes (Kita et al., 1984).

284

285 3.2. Metabolism of marker substrates by CYP6G3

286

287 Thirteen marker substrates were tested for CYP6G3 activity. CYP6G3 showed activity

288 towards the fluorogenic substrate 7-ethoxycoumarin and the colorimetric substrate p-

289 nitroanisole, but no significant activity was seen towards p-nitrophenol, coumarin, 7- 290 benzyloxyresorufin, or with several luminogenic P45MANUSCRIPT0-Glo substrates: luciferin-Me, -CEE, - 291 H, –BE, -PFBE, -ME-EGE or –H-EGE.

292 The rates of CYP6G3-mediated pNAOD and 7ECOD activity were fit well by the

293 Michaelis-Menten equation over the concentration ranges shown in Figure 2. Kinetic

294 constants are shown in Table 1. CYP6G3 pNAOD activity was stimulated by fb 5 with a nearly

295 two-fold increase in k cat and essentially no change in KM (Figure 2).

296 297 3.3. Screening forACCEPTED inhibition of 7ECOD 298

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

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

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

13 ACCEPTED MANUSCRIPT

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

303 significant inhibition of 7ECOD with calculated IC 50 values ranging from 0.23 µM to 26.8

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

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

306 inhibition (data not shown), but the IC 50 value exceeded the highest assayed concentration and

307 could not be estimated accurately.

308

309 3.4. Ligand-binding difference spectra

310

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

312 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 (K S) of 4.3 ± MANUSCRIPT0.3 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 /K S)(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).

320

321 3.5. Metabolism of pesticide substrates by CYP6G3 322 Imidacloprid wasACCEPTED 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

14 ACCEPTED MANUSCRIPT 327 4. Discussion

328

329 4.1. CYP6G3 expression and solubility

330

331 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.

339 340 4.2. Characterization of the activity of the recombinant MANUSCRIPT enzyme towards marker substrates 341

342 To identify marker substrates for characterizing CYP6G3, thirteen potential substrates

343 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). 347 Many P450ACCEPTED enzymes exhibit increased catalytic rates in the presence of cytochrome b 5 348 (Yamazaki et al., 2002). To determine the effect of fb 5 on CYP6G3 activity, pNAOD was

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

350 fold in the presence of fb 5, while the KM was not significantly changed (Fig 2), suggesting an

351 effect on overall turnover but not interactions with substrate. Notably, neither the k cat nor the

15 ACCEPTED MANUSCRIPT

352 KM of CYP6G1-mediated pNAOD was affected by the addition of fb 5 (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 fb 5.

357

358 4.3. Screening for pesticide interactions with CYP6G3

359

360 Oxidative metabolism has been implicated in the resistance of L. cuprina to

361 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, sharesMANUSCRIPT 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.ACCEPTED 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.

375

376

16 ACCEPTED MANUSCRIPT 377 Since 7ECOD activity was the more sensitive of the two marker substrates metabolized

378 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, MANUSCRIPT 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.

395 The organophosphorus insecticides malathion and chlorfenvinphos also inhibited

396 CYP6G3-mediated 7ECOD activity and induced type I binding spectra. Organophosphorus 397 insecticides wereACCEPTED 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 402 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 .

408 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 tr iazineMANUSCRIPT 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).

419 Although imidacloprid is a substrate of CYP6G1 (Fusetto et al., 2017; Hoi et al., 2014;

420 Jou βen 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 metabolicACCEPTED experiments confirmed that CYP6G3 metabolized imidacloprid to the 5- 423 hydroxy and olefin metabolites, both of which are toxic in other (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.

18 ACCEPTED MANUSCRIPT 427 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.

435 A role has been proposed for P450s in diazinon metabolism of the phosphorothioate to

436 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 but MANUSCRIPToxide, 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.

446 447 4.4. ConcludingACCEPTED 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 452 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 .

461

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20 ACCEPTED MANUSCRIPT 462 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.

468

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21 ACCEPTED MANUSCRIPT 469

470 Figure legends

471

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

473

474 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 fb 5.

477

478 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.

480

481 Figure 4. Ligand-induced difference spectra and best fit to the concentration dependence of 482 the difference magnitude for CYP6G3 binding. MANUSCRIPT 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.

485

486 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 ACCEPTEDP450 (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

22 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

496 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).

501

502

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687 Waxman, D.J., Chang, T.K.H., 2006a. Spectrofluorometric analysis of CYP2A6-catalyzed 688 coumarin 7-hydroxylation in: Phillips, I.R., Shepha MANUSCRIPTrd, 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.ACCEPTED 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

31 ACCEPTED MANUSCRIPT 699 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein

700 Express. Purif. 24, 329-337.

701

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32 ACCEPTED MANUSCRIPT Tables

Table 1

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

6G3 pNAOD activity supported by fCPR in the presence and absence of fb 5

a b System Activity kcat KM

6G3 CuOOH 7ECOD 1.7 ± 0.1 1042 ± 96

6G3 fCPR NADPH pNAOD 9.6 ± 0.2 9.8 ± 1.1

6G3 fCPR fb 5 pNAOD 17.8 ± 0.5 13.4 ± 1.9

NADPH a nmol / min / nmol P450 b µM

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33 ACCEPTED MANUSCRIPT Table 2

IC 50 values for the inhibition of CYP6G3 7ECOD activity and spectral binding

parameters for ligand binding to CYP6G3

a b Ligand IC 50 KS Hill ∆Amax ∆Amax /

Coefficient KS endosulfan 0.23 1.4 +/- 0.1 n.a. c 0.064 +/- 0.046

0.001 ketoconazole 5.5 4.3 +/- 0.3 1.6 +/- 0.1 0.019 +/- 0.0044

0.001 chlorfenvinphos 26.8 1.0 +/- 0.1 n.a. 0.056 +/- 0.056

0.001 malathion >100 5.4 +/- 0.4 n.a. 0.030 +/- 0.0056

0.001 DDT >100 28 +/- 1 MANUSCRIPT n.a. 0.046 +/- 0.0016 0.001 lufenuron >100 n.d. d n.a. n.d. n.d. carbaryl >100 n.d. n.a. n.d. n.d. dicyclanil >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 ACCEPTED >100 23 +/- 3 n.a. 0.0167 0.0007 +/-

0.0006 a µM, IC 50 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 d not determined

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ACCEPTED 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.

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

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

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