Comparative Analysis of Recombinant Cytochrome P450 CYP9A61

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Comparative analysis of recombinant cytochrome P450 CYP9A61 from Cydia pomonella expressed in Escherichia coli and Pichia pastoris Xue-Qing Yang, Wei Wang, Xiaoling Tan, Xiao-Qi Wang, and Hui Dong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00372 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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Page 1 of 36

Journal of Agricultural and Food Chemistry

1 2 3 4

Comparative analysis of recombinant cytochrome P450 CYP9A61 from Cydia

5

pomonella expressed in Escherichia coli and Pichia pastoris

6

Xue-Qing Yanga, *, Wei Wang a, Xiao-Ling Tan b, Xiao-Qi Wang a, *, Hui Dong a

7 8 9 10 11 12 13 14 15 16 17 18 19

a

Key Laboratory of Economical and Applied Entomology of Liaoning Province, College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, China b Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China *Corresponding authors. Tel.: +86 02488487148; fax: +86 02488487148 E-mail: [email protected] (X.Q. Yang) [email protected] (X.Q. Wang)

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ACS Paragon Plus Environment


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ABSTRACT Based on prior work, cytochrome P450 CYP9A61 was found to be enriched in fat

41

bodies

and

during

42

lambda-cyhalothrin in Cydia pomonella. In this study, recombinant CYP9A61 was

43

expressed in Escherichia coli and Pichia pastoris, and biochemical properties were

44

investigated. Substrate saturation curves and biochemical properties revealed that,

45

although with the presence of glycosylation, the yeast-secreted CYP9A61 exhibited a

46

higher affinity towards the substrate p-nitroanisole (pNA) and was found to be more

47

stable at certain pH and temperatures than bacterial-produced CYP9A61.

48

Half-inhibitory concentration (IC50) values of three synthetic pyrethroids on both the

49

bacterial and yeast expressed CYP9A61 suggested that recombinant CYP9A61

50

expressed in different hosts exhibits different inhibition properties. Taken together,

51

our findings show that yeast-expressed CYP9A61 exhibits better enzyme activity than

52

is expressed in bacteria, and might be used for further metabolism assays to reveal the

53

insecticide-detoxifying role of CYP9A61 in C. pomonella.

54

KEYWORDS:

55

Biochemical properties, Inhibition properties

Cydia

feeding

stages,

pomonella

and

(L.),

transcription

P450,

was

Detoxification,

56 57 58 59 60 61 2


induced

by

Pyrethroids;

Page 3 of 36


62

INTRODUCTION

63

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), an

64

economically important orchard pest of apples and pears worldwide 1, 2, has developed

65

resistance to several different classes of insecticides, including synthetic pyrethroids

66

(Pys)

67

(IGRs) 2. Cytochrome P450 (P450 or CYP) enzymes are important heme-containing

68

monooxygenases that exist in almost all organisms. In insects, P450s are focused on

69

due to their detoxification role and are responsible for insecticide resistance

70

Previous studies focused on the underlying resistance mechanisms of the insecticide

71

resistance in field populations of C. pomonella have indicated that a P450-based

72

metabolic resistance is one of the main mechanisms 1, 2, 5, 13.

3, 4

, organophosphates (OPs)

5-7

, neonicotinoids

5, 8

and insect growth regulators

9-12

.

73

Most recently, the CYP9A61 transcripts were found to be more abundant in the

74

silk gland and fat body than in other tissues and in feeding stages compared to

75

non-feeding stages. Transcript of CYP9A61 and p-nitroanisole-O-dealkylation

76

(pNAOD)

77

chlorpyrifos-ethyl, empirically demonstrating that this P450 is potentially involved in

78

the insecticide-detoxifying process

79

pNAOD activity, there is no direct evidence that CYP9A61 detoxifies insecticides. To

80

further elucidate the insecticide-detoxifying role of CYP9A61, a more rigorous

81

assessment of the biochemical properties of CYP9A61, as well as the interactions of

82

CYP9A61 with insecticides in an in vitro assay system should be performed. However,

83

such work has been hindered due to the lack of enough (approximately a milligram)

activity

were

significantly

induced

by

lambda-cyhalothrin

and

14

. Apart from the induction of CYP9A61 and

3



Page 4 of 36

84

active form of pure CYP9A61 with high enough activity for biochemical study. It is

85

known that characterization of a recombinant P450 is really difficult since CYPs are

86

one of most challenging enzymes to functionally characterize due to the difficulty of

87

recombinantly expressing these membrane-associated monooxygenases.

88

Currently, purification of the native enzyme of CYP from insect tissues was

89

technically difficult, time-consuming, and gave only a very low yield due to the very

90

low CYPs content in insects 15. Another way to obtain a large amount of protein is to

91

express the protein in heterologous expression systems using recombinant DNA

92

technology. Currently, both prokaryotic and eukaryotic microorganism expression

93

systems have been frequently selected as expression hosts. To our knowledge, insect

94

P450s from various sources have been cloned and functionally expressed in

95

recombinant baculovirus-infected Sf9 insect cells 11, 16, Escherichia coli

96

yeast

97

technically more complicated and produces a relatively low yield of protein. The yeast

98

and

99

baculovirus-expression system since they are easy to handle, produce a high yield,

100

and are cost-effective 15. Recently, some insect and mite CYPs have been functionally

101

co-expressed with NADPH P450 reductase (CPR) in E. coli 18, 21, and there are also a

102

few insect CYPs that have been functionally expressed in prokaryotic systems without

103

co-expression with CPR 15, 17. However, functional expression and characterization of

104

CYPs in C. pomonella has not kept pace with that in other pests and mites 12. Thus,

105

the knowledge of expression of the CYP9A61 in a heterologous system and the

15, 17, 18

and

19, 20

. Among these expression systems, the baculovirus-expression system is

E.

coli

expression

systems

have

advantages

4


compared

with

the

Page 5 of 36


106

enzyme properties of the produced protein, as well as the inhibitory activity of Pys

107

against CYP9A61, may provide a better understanding of insecticide detoxification

108

mechanisms in this species.

109

Taking into account the significance of this P450, further research was initiated

110

toward the functional overexpression of C. pomonella CYP9A61 in E. coli. and P.

111

pastoris, including determining the purification, catalytic activity, and inhibition

112

properties of the enzymes using three widespread commercially-used synthetic

113

pyrethroids. These results will then provide information for future investigation of the

114

roles of CYP9A61 in detoxification of insecticides.

115

MATERIALS AND METHODS

116

Strains, plasmids, and chemicals. The P. pastoris strain X-33 (wild-type) and the

117

expression vector pPICZαB were purchased from Invitrogen (CA, USA). The

118

pET-32a (+) plasmid was obtained from Novagen (Heidelberg, Germany). The E. coli

119

strain DH5α, the Ex Taq DNA polymerase, and restriction endonucleases were

120

purchased from Takara (Dalian, China) and were used as specified by the suppliers.

121

Yeast nitrogen base (YNB, without amino acids and ammonium sulfate) was obtained

122

from Becton, Dickinson and Company (USA). The E. coli DH5α cells harbouring the

123

pPICZαB plasmid were cultured at 37℃ in Luria-Bertani (LB) medium containing 25

124

µg/ml Zeocin (Invitrogen, USA). The p-nitroanisole, p-nitrophenol, analytical grade

125

insecticides, and the P450 inhibitor quercetin

126

(St. Louis, MO). All other chemicals and reagents (analytical grade) were

127

commercially available.

22

were purchased from Sigma-Aldrich

5



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128

Isolation of CYP9A61 and construction of the prokaryotic expression vector. The

129

total RNA was extracted from five third-instar C. pomonella larvae using the RNAiso

130

Plus Kit (Takara) based on the manufacturer's instructions, and was then treated with

131

DNase I (MBI, Fermentas) to remove any genomic DNA contamination. A total of

132

1µg RNA was used to synthetize first-strand cDNA by using the RevertAid™ First

133

Strand cDNA Synthesis Kit (MBI) as described by the manufacturer. The open

134

reading frame (ORF) with truncation of the N-terminal transmembrane domain

135

CYP9A61 was amplified by RT-PCR using F1 and R1 (Table 1) as the primer pair.

136

The PCR product was cleaned using the Biospin Gel Extraction Kit (Bioer

137

Technology Co., Ltd., Hangzhou, China) as recommended by the manufacturer, and

138

was then digested with KpnⅠ and Not Ⅰrestriction enzymes. Digestion products

139

were ligated into the expression vector pET-32a (+) already digested with the same

140

restriction enzymes to create the expression plasmid CYP9A61-pET 32a (+). In order

141

to facilitate purification, a 6×His tag was fused on the C-terminal of CYP9A61; this

142

was then inserted into the plasmid pET-32a (+) to create pET-32a (+)-CYP9A61 (Fig.

143

S1a). The recombinant pET-32a (+)-CYP9A61 plasmid was then transformed into E.

144

coli BL21 (DE3). Three clones were sequence-verified by Shanghai Sunny Biotech

145

Co., Ltd., China.

146

Expression and purification of recombinant CYP9A61 in E. coli. Single positive

147

colonies were inoculated into 1 L Luria-Bertani (LB) medium containing 1%

148

casamino acids, 17 mM KH2PO4 and 72 mM K2HPO4, 100µg/ml ampicillin with

149

shaking at 220

rpm at 37 ℃

until OD600=0.6. 6


After that,

14

of

isopropyl

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150

β-D-thiogalactopyranoside (IPTG) at final concentration of 0.2 mM was added, and

151

cells were grown at 18℃ with shaking at 220 rpm for 48 h. Cells were harvested by

152

centrifugation at 4℃ (12,000g, 20 min), and the cell pellets were resuspended in 20

153

ml of TA buffer (50 mM Tris·acetate, pH 7.6, containing 250 mM sucrose and 0.25 M

154

EDTA) containing 0.25 mg/ml lysozyme. This mixture was shaken at 4℃ for 1 h at

155

80 rpm. Then the membrane fraction was isolated as described by Ding et al. 17. The

156

crude samples were applied to a Ni-NTA agarose gel column (Transgen, Beijing,

157

China), and the recombinant CYP9A61 was purified as described by the manufacturer.

158

The purified CYP9A61 was analyzed by sodium dodecyl sulfate polyacrylamide gel

159

electrophoresis (SDS-PAGE) and stained with Coomassie blue G-250 (Roche).

160

Construction of yeast expression vector. The PCR products and the expression

161

vector pPICZαB were digested and ligated as described above. In order to facilitate

162

purification, a 6×His tag was fused on the C-terminal of CYP9A61, and was then

163

inserted into the plasmid pPICZαB to create CYP9A61-pPICZαB (Fig. S1b). The

164

recombinant pPICZαB vector was transformed into E. coli DH5α competent cells, and

165

colonies were then screened on LB solid medium containing 25 µg/ml Zeocin to

166

obtain positive recombinant colonies containing CYP9A61-pPICZαB. Restriction

167

analysis and sequencing was further performed to verify the CYP9A61-pPICZαB

168

expression construct sequences. Subsequently, recombinant CYP9A61-pPICZαB

169

plasmid was linearized with SalⅠ, and the products were electroporated into

170

competent P. pastoris X-33 cells. The empty vector control was used as a negative

171

control. The positive transformants were screened on YPD plates [2% (w/v) tryptone, 7



172

1% (w/v) yeast extract, 2% (w/v) glucose, 2% (w/v) agar] containing 100 µg/ml

173

Zeocin. The recombinant yeast genomic DNA was isolated and the presence of

174

inserted CYP9A61 was further verified by PCR using genomic DNA as templates,

175

and using 5′AOX-F coupled with α-factor R, and 5′AOX-F coupled with 3′AOX-R as

176

the primer pair (Table 1). As shown in Fig. S1c, two transformants were PCR

177

confirmed for harboring the CYP9A61.

178

Expression and purification of recombinant CYP9A61 in P. pastoris.

179

PCR-confirmed transformants were grown in 20 mL buffered complex glycerol media

180

[BMGY, composed of 1% (w/v) yeast extract, 2% (w/v) tryptone, 1.34% YNB, 1%

181

glycerol, 0.2 µg/ml biotin, buffered with 0.1 M potassium phosphate, pH 6.0] at 30℃

182

at 200 rpm until the cultures had reached an OD600 of 2~6. The cells were harvested

183

by centrifugation at 3,000×g for 5 min, resuspended in 200 mL buffered methanol

184

complex medium (BMMY; 1% yeast extract, 2% tryptone, 1.34% YNB, 1% methanol,

185

0.2 µg/ml biotin, pH 6.0) to an OD600 of 1.0 on a shaker. Methanol was added into the

186

culture to a final concentration of 1% (v/v) every 24 h to maintain the induction. A

187

negative control containing the pPICZαB plasmid without any exogenous gene

188

inserted was carried out in parallel. For analyzing of the expression, 1 ml of culture

189

was taken at 24 h intervals. After 72 h methanol induction, the supernatant was

190

harvested and the proteins were precipitated at 4℃ by adding ammonium sulfate to a

191

final concentration of 80% (w/v) as described by the protein purification handbook

192

(Biotech, Amersham Pharmacia, 2001). The precipitated proteins were collected by

193

centrifugation at 4℃ at 100,000×g for 20 min, dissolved in 0.1 M sodium phosphate 8


Page 8 of 36

Page 9 of 36


194

buffer (pH 7.8), and followed by purification by Ni-NTA agarose gel column

195

(Transgen, Beijing, China).

196

To determine if the bands that appeared on SDS-PAGE corresponded with the

197

target protein, a Western blot analysis was performed using an anti-His antibody.

198

Equivalent proteins (10 µl) of methanol-induced supernatant of the empty vector

199

control for 72 h, and methanol-induced supernatant of positive transformants at 24

200

and 72 h were separated by 12% SDS-PAGE and transferred to a nitrocellulose (NC)

201

membrane by a Bio-Rad Trans-Blot SD (Hercules, USA). Subsequently, the NC

202

membrane was treated with 5% skimmed milk in Tris-HCl buffer containing 0.5%

203

Tween 20 (TBST) for 2 h, and was then incubated in antisera against His tag antibody

204

at a dilution of 1 : 5000. Thereafter, goat-anti-mouse antibody at a dilution of 1 : 3000

205

was incubated with the NC membrane, and the immunoreaction bands were detected

206

using an Enhanced Chemluminescent Kit (Boster, Wuhan, China) as described by the

207

manufacturer.

208

Both the recombinant proteins produced from E. coli and P. pastoris were

209

dialyzed against 0.1 M sodium phosphate buffer (pH 7.8) on a magnetic stirrer at 4℃

210

overnight. The dialysis buffer was changed three times during dialysis. The dialyzed

211

recombinant CYP9A61 was incubated for 36 h at 4 °C with enterokinase (Wuhan

212

More Biotechnology Co., Ltd., China) to generate Trx-free CYP9A61. To maintain an

213

active protein, glycerol was added to a final concentration of 20%, and the sample

214

was then flash frozen in liquid nitrogen and stored in aliquots at -80℃ before use. The

215

protein concentration was determined by the Bradford assay using BSA as a standard 9



216

23

217

according to the method of Omura and Sato 24.

. CYP9A61 concentrations were determined by reduced vs. oxidised spectroscopy

218

Deglycosylation assay was carried out using 20 µg purified protein expressed in

219

yeast with the Enzymatic Protein Deglycosylation Kit (Saint Louis, MO, USA) from

220

Sigma. After that, the sample was heated for 5 min at 95℃ and the molecular mass of

221

the deglycosylated protein was determined by SDS-PAGE using a 12% gel.

222

p-nitroanisole-O-dealkylation (pNAOD) activity of recombinant CYP9A61. The

223

p-nitroanisole-O-dealkylation (pNAOD) activity was determined in a final volume of

224

200 µl with chromogenic substrate p-nitroanisole (pNA) as described previously 25, 26

225

with slight modification. In brief, 1 nM recombinant CYP9A61 was reconstituted

226

with 0.03 nM housefly P450 reductase

227

buffered with 2 mM pNA on Nunc 96-well transparent microplates (Nunc, Roskilde,

228

Denmark). Subsequently, plates were pre-incubated for 5 min at 30 °C before

229

reactions were initiated by addition of 10 µl of 9.6 mM reduced form of nicotinamide

230

adenine dinucleotide phosphate (NADPH). After 1 h of reaction, the assay was

231

quenched with 10 µl of 2 M NaOH and absorbance was read in an Infinite M200 PRO

232

Microplate Reader (Tecan, Switzerland) at 405 nm to monitor the generation of

233

p-nitrophenol (pNP) from pNA. The activity was expressed as nmole pNP min-1 mg of

234

protein-1. Three replicates of control reactions (without NADPH addition) were run

235

for each P450 source. To determine the steady-state kinetic parameters Vmax and Km,

236

various concentrations (1000, 500, 100, 80, 60, 40, 20, 10, 4 and 2 µM) of pNA were

237

used. The kinetic parameters were determined from the double reciprocal

27

in 0.1 M sodium phosphate (pH 7.8)

10


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238

Lineweaver–Burk plots.

239

Inhibition studies. The inhibition of pNAOD by various insecticides was measured

240

using the 96-well microplate in the presence of pNA (10 µM), 0.1 M sodium

241

phosphate buffer (pH 7.8), 1.0 nM CYP9A61, 0.03 nM housefly P450 reductase, and

242

different concentrations (200, 20, 2, 0.2, 0.02 and 0.002 µM) of insecticides in a final

243

volume of 190 µl. After incubation for 5 min at 30 ℃, reaction was initiated by

244

adding 10 µl of 9.6 mM NADPH. Reactions were run for 1 h, and the absorbance was

245

determined at 405 nm. Insecticide stocks were dissolved and diluted in acetone, and a

246

solvent control was included to correct for any solvent effects across the dilution

247

range. Quercetin, a potent P450 inhibitor

248

addition, a control reaction (no NADPH) was also conducted. IC50 values were

249

calculated using GraphPad Prism 5 (San Diego, USA) using the following equation 26,

250

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

251

Effects of temperature and pH on pNAOD activity. The effect of pH on enzyme

252

activity was analyzed by incubating aliquots of CYP9A61 protein at 30℃ using 0.1 M

253

sodium phosphate buffer at different pH (5.5–10.0). The activity of CYP9A61

254

measured at pH 7.8 was considered to be 100 %.

22

, was employed as a positive control. In

255

The effect of temperature on enzyme activity was determined by incubating

256

aliquots of CYP9A61 protein at various temperatures (15–55 ℃) in 0.1 M sodium

257

phosphate (pH 7.8). After 1 h of reaction, the assay was quenched with 10 µl of 2 M

258

NaOH and absorbance was read as described above. The activity of CYP9A61

11



259

analyzed at 30℃ was regarded as 100 %. The effect of temperature on enzyme

260

stability was investigated by pre-incubating the purified CYP9A61 at various

261

temperatures (15–55 ℃) in 0.1 M sodium phosphate (pH 7.8) for 5 min. Subsequently,

262

10 µl of 9.6 mM NADPH was added to initiate the reaction at 30 ℃. After 1 h of

263

reaction, the assay was quenched with 10 µl of 2 M NaOH and absorbance was read

264

as described above. The relative activity was determined and the activity of CYP9A61

265

stored at -80 ℃was regarded as 100 %.

266

Structural modeling for CYP9A61. The 3D structure of CYP9A61 was modeled

267

using the SWISS-MODEL server (http://swissmodel.expasy.org). The target template

268

sequence was searched with BLAST against the primary amino acid sequence

269

contained in the SWISS-MODEL template library. A total of 295 templates were

270

found. For each identified template, the template's quality was predicted from features

271

of the target-template alignment. The templates with the highest quality were selected

272

for model building. As a result, the crystal structure of Cytochrome P450 3A4 (PDB

273

code 3nxu.1, chain A, resolution 2.0Ǻ) from the human was selected as a template

274

after searching for the PDB database. Based on the model obtained, the 3D structure

275

of CYP9A61 was generated using the Swiss-PdbViewer program, version 4.1.0. The

276

reliability of 3D model was validated using Profile-3D 28.

277

Statistical analysis. Statistical analysis was conducted using SPSS 12.0 (IBM,

278

Chicago, USA). The results are shown in mean of triplicates ± standard deviation (SD)

279

and were plotted using GraphPad Prism 5 (San Diego, USA).

12


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280

RESULTS

281

Expression of recombinant CYP9A61 in E. coli. The ORF of CYP9A61 encodes a

282

protein of 515 amino acids, with truncation of N-terminal transmembrane domain at

283

residues 2-24 14, was isolated by PCR. The CYP9A61 expressed in E. coli was mainly

284

produced as inclusion bodies, however, the 12 % SDS-PAGE (5 µl protein loaded)

285

showed that the recombinant CYP9A61 produced a low amount of soluble P450 (Fig.

286

1a, Lane 1). Additionally, the recombinant CYP9A61 exhibited an expected molecular

287

mass of approximately 78 kDa (Fig. 1a, Lane 1), which is the molecular weight of the

288

CYP9A61 holoenzyme (62.04 kDa) plus the Trx-tag (~13 kDa) and His-Tag (~1.0

289

kDa) and S-Tag (~1.7 kDa) in the pET-32a (+) plasmid, and the fused 6×His tag (~1.0

290

kDa) at the C-terminal of CYP9A61. After cleaved by recombinant enterokinase, a

291

single band with estimated molecular weight of 62 kDa was obtained (Fig. S2).

292

Expression of CYP9A61 in P. pastoris. The 12 % SDS-PAGE showed that the

293

recombinant CYP9A61 expressed a band

294

(approximately 80 kDa) than expected (63 kDa, 62.04 kDa for the enzyme and ~1kDa

295

for the His-tag), and the yield increased as induction time progressed from 24 h to 72

296

h (Fig. 1b). Thus, the optimal induction time was determined at 72 h post methanol

297

induction. The target band was not observed in non-CYP9A61 inserted control

298

(pPICZαB transformed into X-33) (Fig. 1b, Lane 6). Western blot analysis

299

provided further evidence that the recombinant CYP9A61 expressed in yeast with

300

higher molecular mass than expected size (Fig. S3).

with higher molecular mass

13



301

Purification of recombinant CYP9A61. The soluble recombinant CYP9A61 was

302

purified in E. coli (Fig. 1a) and P. pastoris (Fig. 1b) by affinity chromatography using

303

Ni2+-NTA resin (Transgen, Beijing, China). After purification, approximately 1.02

304

mg/ml ± 0.251 (n=3) and 4.086 mg/ml ± 0.985 (n=3) of total recombinant protein

305

were obtained in 1 L and 500 mL culture expressed in E. coli (E-CYP9A61) and P.

306

pastoris (P-CYP9A61), respectively.

307

The CO-difference spectrum indicated that the CYP9A61 was expressed

308

predominantly as P450, with only a small amount of P420 both in E. coli (Fig. S4a)

309

and P. pastoris (Fig. S4b),suggesting that the purified recombinant CYP9A61 is of

310

good quality

311

pastoris

312

nmol/mg protein ± 0.185 (n=3), respectively.

24

. Results indicated that the CYP9A61 expressed in E. coli and P.

with P450 content of 3.669 nmol/mg protein ± 0.842 (n=3) and 0.646

313

Deglycosylation assay was further carried out to determine the potential

314

glycosylation of yeast-produced recombinant CYP9A61 using PNGaseF (Fig. S5).

315

The PNGaseF-treated sample exhibited two bands on the gel, one was completely

316

deglycosylated (~62 kDa), and the other band represent incompletely deglycosylated

317

CYP9A61 (>62 kDa), suggesting the presence of glycosylation in yeast-produced

318

recombinant CYP9A61.

319

p-nitroanisole-O-dealkylation activity (pNAOD) assay. The pNAOD activity of

320

recombinant CYP9A61 expressed in E. coli. BL21 (DE3) and P. pastoris X-33 against

321

pNA was determined. The recombinant E-CYP9A61 exhibited an activity value of

322

0.2512 ± 0.068 and 0.2685 ± 0.075 nmol of pNP/min/mg protein respectively, for 14


Page 14 of 36

Page 15 of 36


323

Trx-contained and Trx-free CYP9A61, whereas the value for recombinant

324

P-CYP9A61 is 87.586 ± 9.54 nmol of pNP/min/mg protein. The pNAOD activity was

325

un-detectable in non-CYP9A61 transformed P. pastoris X-33 cell after methanol

326

induction.

327

The pNAOD activity of recombinant CYP9A61 displayed Michaelise-Menten

328

kinetics over the pNA substrate range shown in Fig. 2. The E-CYP9A61 (Fig. 2a) and

329

P-CYP9A61 (Fig. 2b) exhibited Vmax values of 4.950±0.316 and 91.78±4.17 nmol of

330

pNP/min/mg protein, and with Km values of 65.20±13.2 and 36.19±6.12 µM against

331

pNA, respectively.

332

Inhibition study. To further characterize the E-CYP9A61 and P-CYP9A61, three Pys

333

(cypermethrin, permethrin and lambda-cyhalothrin), and the potent P450 inhibitor

334

quercetin were selected to compare their inhibition profile against CYP9A61 in two

335

different microbial expression hosts (Table 2). A clear difference of inhibition

336

properties exists between E-CYP9A61 and P-CYP9A61 as demonstrated by the IC50

337

values of all tested molecules (Table 2). In details, the cypermethrin showed IC50

338

values of 27.8 and 15.8 µM for E-CYP9A61 and P-CYP9A61, respectively; whereas

339

permethrin exhibited IC50 values of 78.9 µM for E-CYP9A61 and 41.2 µM for

340

P-CYP9A61, and the lambda-cyhalothrin with a IC50 values of 19.7 µM for

341

E-CYP9A61 and 10.3 µM for P-CYP9A61 (Table 2). Strongest inhibition was

342

observed by the quercetin with an IC50 value of 0.4 and 0.2 µM for E-CYP9A61 and

343

P-CYP9A61, respectively (Table 2).

15



344

Effect of pH and temperature on CYP9A61 activity. Both the E-CYP9A61 and

345

P-CYP9A61 exhibited maximum activity at pH 7.5 and 8.0, and activity decreased

346

above pH 9.0 or below pH 7.0. Moreover, the E-CYP9A61 was more rapidly

347

inactivated below pH 7.0 (Fig. 3a).

348

The E-CYP9A61 was most active at 30℃ whereas the P-CYP9A61 showed

349

maximum activity at 30℃ and 35℃. Both the E-CYP9A61 and P-CYP9A61 were

350

rapidly inactivated at elevated temperatures. The P-CYP9A61 exhibited higher

351

activity than the E-CYP9A61 at most determined temperatures (Fig. 3b). The activity

352

of E-CYP9A61 and P-CYP9A61 decreased after incubation at 25°C or 30°C.

353

However, both of the enzymes were stable (> 80% remaining active) when kept below

354

35℃ for 1 h at pH 7.8, and the activity dropped dramatically and retained ~20%

355

residual activity at 50 and 55℃. The P-CYP9A61 exhibited higher thermostability

356

than E-CYP9A61 at 35 and 40℃ (Fig. 3c).

357

Structural analysis of CYP9A61. The structure of the CYP9A61 3D model is shown

358

in Fig. S6. The CYP9A61 contains 16 α-helices and 9 β-sheets connected by loops.

359

The CYP9A61 shares a 34.62 % amino acid sequence identity match with the human

360

CYP3A4 (PDB: 3nxu.1A). The amino acid residues which are a formation of the

361

cytochrome P450 heme-binding domain FxxGxxxCxG (Phe 473, Gly 474, Leu 475,

362

Gly 476, Pro 477, Arg 478, Asn 479, Cys 480, Ile 484, Gly 482) are shown in red (Fig.

363

S6). The result of Profile-3D shows the residues with an average 3D−1D score >0.05

364

(Fig. S7), indicating that the 3D model is reasonable.

16


Page 16 of 36

Page 17 of 36


365

DISCUSSION

366

A previous study documented that cytochrome P450 CYP9A61 was associated with

367

lambda-cyhalothrin detoxification in C. pomonella

368

insecticide detoxification role of CYP9A61, we strove to functionally express the

369

recombinant CYP9A61 in bacteria and yeast with truncation of the N-terminal

370

transmembrane domain. Successful expression of CYP9A61 apoprotein was achieved

371

in this study.

14

. To further investigate the

372

The molecular mass of the secreted recombinant CYP9A61 in P. pastoris

373

(approximately 80 kDa) was higher than that expressed in E. coli (62.04 kDa, without

374

the Tags). The possible reason might be glycosylation, one of the most common

375

post-translation modifications occurring in P. pastoris cells

376

N-glycosylation prediction web tool (http://www.cbs.dtu.dk/services/NetNGlyc/),

377

three potential N-glycosylated sites, including NYTT (residues 3-6), NLTF (residues

378

196-199) and NKTV (residues 291-294) were predicted. The potential glycosylation

379

in yeast-produced recombinant CYP9A61 was further confirmed by treatment with

380

PNGaseF (Fig. S5). Our finding is in line with Toxoplasma gondii surface antigen 2

381

gene SAG2

382

pastoris. Unlike E. coli, the P. pastoris is capable of conducting many of the

383

post-translational modifications which are usually performed in higher eukaryotes,

384

such as correct folding, N-linked glycosylation, disulphide bond formation,

385

proteolytic processing and processing of signal sequences

386

not shown) confirmed that the N-glycosylated CYP9A61 retained catalytic activity

30

and mammalian acetylcholinesterase

31

29

. Using the

heterologously expressed in P.

17


32

. Further analysis (data


Page 18 of 36

387

(using pNA as substrate); this result is in line with previous characterization of P450s

388

from other sources 33, 34.

389

Most of the recombinant CYP9A61 expressed in E. coli as inclusion bodies.

390

Although the recombinant CYP9A61 produced high quantities of P450 (~3.669

391

nmol/mg protein), which is much higher than CYP9Js from Aedes aegypti

392

soluble protein level is relative low. It has been shown that fusing the MBP signal

393

sequence increases the yield of soluble fusion protein in the cytoplasm after being

394

fused on the N-terminus of the native protein

395

sequence provides an E. coli-derived TIR and enables the translocation of expressed

396

protein into the disulfide bond formation promoting environment of the periplasm 31.

397

Thus, further studies introducing the MBP or OmpA sequence are required for

398

improving the yield of soluble protein levels of CYP9A61 in E. coli.

35

, the

36

. Furthermore, the OmpA leader

399

For the yeast expression system, the prepro leader sequence of the α-factor of

400

pPICZαB could direct the secretion of active CYP9A61 into the medium. The amount

401

of P450 was 0.646 nmol/mg protein, which was much higher than CYP6D1 from the

402

house fly (Musca domestica) expressed in another yeast cell line, Saccharomyces

403

cerevisiae by culture in media with glucose using expression vector pYES2 19. In this

404

study, the pNAOD activity of yeast-produced recombinant CYP9A61 was 87.586 ±

405

9.54 nmol pNP min/mg protein against pNA, which was much higher than the

406

recombinant CYP9A61 expressed in E. coli. The pNAOD activity of recombinant

407

CYP9A61 in this study was also higher than recombinant CYP9A12 and CYP9A14

408

from Helicoverpa armigera expressed in S. cerevisiae without fusion CPR or 18


Page 19 of 36


409

introducing any signal sequence or tag, which exhibited pNAOD activity values of

410

0.59 and 0.42 nmol pNP min/mg protein against pNA

411

pNAOD activity of recombinant CYP9A12 and CYP9A14 was determined in yeast

412

cell lysate, not with purified enzyme, so that reduces the values for comparison of the

413

pNAOD activities of CYP9A12 and CYP9A14 with CYP9A61.

20

. It is noteworthy that the

414

The optimal pH of the secreted recombinant CYP9A61 was around 7.5, which is

415

similar to those of E. coli-produced enzyme. This optimal pH is in line with total P450

416

from midgut microsomes of the black swallowtail, Papilio polyxenes 37. The enzyme

417

expressed in E. coli is more rapidly inactivated by decreased pH (below pH 7.0),

418

which might be the lack of glycosylation. The optimal temperature of the secreted

419

recombinant CYP9A61 was observed at 30℃ and 35℃, whereas the maximum

420

activity of CYP9A61 expressed in E. coli was determined to be 30℃.

421

To gain a better understanding the role of CYP9A61 in detoxification of

422

insecticides, we investigated the interactions of recombinant CYP9A61 with

423

pyrethroids. Both the E-CYP9A61 and E-CYP9A61 were rapidly inhibited by the

424

insecticides cypermethrin, permethrin and lambda-cyhalothrin. However, the

425

inhibitory effect of all test Pys against secreted recombinant CYP9A61 was ~2-fold

426

higher than E-CYP9A61. One possible explanation for such differences is the lack of

427

post-translational

428

disulphide bond formation and proteolytic processing in E. coli influence the

429

properties of a eukaryote’s protein. The IC50 values for insecticides determined in the

430

current study against CYP9A61 are significantly higher than those against Anopheles

modifications---for

instance

correct

19


folding,

glycosylation,


22

431

gambiae CYP6Z2

; this could be a result of different enzyme sources. In general,

432

different enzymes from different organisms can be expected to show different

433

biochemical properties. Another possible explanation for such a difference is that

434

fluorescent substrate benzyloxyresorufin may have a higher specificity than pNA used

435

in our study. But in another study of Drosophila melanogaster CYP6G1 expressed in

436

E. coli, the pNA was found to be a suitable substrate for high-throughput studies 26.

437

Thus the substrate spectrum of CYP9A61 should be screened in further research.

438

Differences between E-CYP9A61 and P-CYP9A61 were also observed in the

439

thermostability and Km value. The P-CYP9A61 was more thermostable than

440

E-CYP9A61 because of the lack of robust post-translational processing in the

441

prokaryotic expression system and the biological activity of produced recombinant

442

proteins are always low

443

smaller than those of E-CYP9A61, suggesting that P-CYP9A61 has higher affinity

444

with pNA. This is likely a better reflection of the situation of the natural enzyme.

445

Previous research suggested that pyrethroid compounds were metabolized by P450s

446

including CYP6 isoforms

447

insecticide-detoxifying role of the CYP9A61 in C. pomonella, however, there is still a

448

long way to go. At least, it requires investigation of turnover of the pesticides in

449

question by CYP9A61 and analysis of the metabolites produced.

38

. However, the Km value of P-CYP9A61 against pNA was

39

, CYP9, CYP321, and CYP337

40

. To investigate the

450

This present study documents the high-level heterologous expression and

451

biochemical characteristics of the recombinant CYP9A61 expressed in E. coli and P.

452

pastoris. Results suggested that the yeast-expressed CYP9A61 had higher affinity 20


Page 20 of 36

Page 21 of 36


453

towards the substrate pNA and different inhibition properties, and was found to be

454

more stable than that expressed in bacteria at some pH and temperature conditions.

455

Further functional studies, especially metabolic tests, should be performed to find out

456

the precise role of CYP9A61 in detoxification of insecticides.

457 458

■ASSOCIATED CONTENT

459

Supporting Information Available: Supplements to the schematic diagram depicting

460

the construction of the recombinant plasmids (Figure S1), western blot analysis of

461

recombinant CYP9A61 (Figure S2), Fe2+-CO vs. Fe2+ difference spectrum (Figure S3),

462

SDS-PAGE of enzymatically deglycosylated recombinant CYP9A61 expressed in

463

yeast (Figure S4), the tertiary structure of CYP9A61 (Figure S5) and Profile 3D score

464

of the CYP9A61 model (Figure S6) are contained in supporting 383 information. The

465

Supporting Information is available free of charge

466

http://pubs.acs.org.

467

AUTHOR INFORMATION

468

Corresponding Author

469

*Tel.: +86 02488487148. Fax: +86 02488487148. E-mail: [email protected]

470

(X.Q. Yang); [email protected] (X.Q. Wang).

471

Notes

472

The authors declare no competing financial interest.

473

ACKNOWLEDGEMENTS

474

We thank Dr. Pei-Wen Qin (College of Plant Protection, Shenyang Agricultural 21


via the Internet at


475

University) for drawing the chemical formula of pyrethroids, and also thank John

476

Richard Schrock from Emporia State University (USA) for proofreading an earlier

477

version of this manuscript. This research was supported by the Scientific Research

478

Foundation of Talent Introduction of Shenyang Agricultural University (Grant

479

20153011), and was supported in part by National Natural Science Foundation of

480

China (Grant 31501666) and General Project of Education Department of Liaoning

481

Province (Grant L5015489).

482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 22


Page 22 of 36

Page 23 of 36


497

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Helicoverpa armigera from Asia. Insect. Biochem. Molec. 2004, 34, 763–773.

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Daborn, P.J.; Russell, R.J.; Gillam, E.M.J.; Oakeshott, J.G. Soluble and

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membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia

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acetylcholinesterase, expressed and reconstituted from Escherichia coli.

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protein production using the Pichia pastoris expression system. Yeast 2005, 22,

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Mkrtchian, S.; Ingelman-Sundberg, M. Colorectal cancer-specific cytochrome

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Core glycosylation of cytochrome P-450 (arom). Evidence for localization of N

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terminus of microsomal cytochrome P-450 in the lumen. J. Biol. Chem. 1993, 27



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(36) Stevenson, B.J.; Pignatelli, P.; Nikou, D.; Paine, M.J.I. Pinpointing P450s

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associated with pyrethroid metabolism in the dengue vector, Aedes aegypti:

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developing new tools to combat insecticide resistance. PLoS Neglect. Trop. D.

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2012, 6, e1595.

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(36) Kapust, R.B.; Waugh, D.S. Escherichia coli maltose-binding protein is

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uncommonly effective at promoting the solubility of polypeptides to which it is

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fused. Protein Sci. 1999, 8, 1668–1674.

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(37) Cohen, M.B.; Berenbaum, M.R.; Schuler, M.A. Induction of cytochrome

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P450-mediated detoxification of xanthotoxin in the black swallowtail. J. Chem.

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Ecol. 1989, 15, 2347–2355.

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(38) Lico, C.; Chen, Q.; Santi, L. Viral vectors for production of recombinant proteins in plants. J. Cell. Physiol. 2008, 216, 366–377.

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(39) Duangkaew, P.; Pethuan, S.; Kaewpa, D.; Boonsuepsakul, S.; Sarapusit, S.;

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Rongnoparut, P. Characterization of mosquito CYP6P7 and CYP6AA3:

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differences in substrate preference and kinetic properties. Arch. Insect. Biochem.

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Physiol. 2011, 76, 236–248.

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(40) Joußen, N.; Agnolet, S.; Lorenz, S.; Schöne, S.E.; Ellinger, R.; Schneider, B.;

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Heckel, D.G. Resistance of Australian Helicoverpa armigera to fenvalerate is

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due to the chimeric P450 enzyme CYP337B3. Proc. Natl. Acad. Sci. USA 2012,

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628 28


Page 28 of 36

Page 29 of 36


629

Figure Legends

630

Figure 1. Expression and purification of recombinant CYP9A61 protein in

631

Escherichia coli (a) and Pichia pastoris (b). The samples (5 µl) were separated on 12 %

632

SDS-PAGE gels and were coomassie blue stained. (a) Lane 1: eluted proteins using

633

elution buffer containing 100 mM imidazole; Lane 2 and 3: recombinant CYP9A61

634

with (Lane 2) or without (Lane 3) induced by 0.2 mM IPTG before loading onto

635

Ni2+-NTA column. (b) Lane 1: Un-induced supernatant of positive transformant at 72

636

h; Lanes 2-4: methanol induced supernatant of positive transformant at 24, 48 and 72

637

h, respectively; Lane 5: methanol induced supernatant of control transformant

638

(pPICZαB-X-33) at 72 h; Lane 6: purified CYP9A61 in P. pastoris. M: The molecular

639

weight markers from top to bottom are β-galactosidase (116.0 kDa), bovine serum

640

albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), and

641

REase Bsp98I (25.0 kDa).

642

Figure 2. Substrate saturation curves for p-nitroanisole O-dealkylation (pNAOD) by

643

CYP9A61 expressed in Escherichia coli (a) and Pichia pastoris (b). Reactions were

644

performed using a 96-well microplate. Reaction systems (200 µl) contained various

645

concentrations of pNA (1000, 500, 200, 100, 80, 60, 40, 20, 10, 4 and 2 µM), 10 µl of

646

CYP9A61. Plates were pre-incubated for 5 min at 30 °C before reactions were

647

initiated by addition of 10 µl of 9.6 mM reduced form of NADPH. After 1 h, the assay

648

was quenched with 10ml of 2 M NaOH and absorbance was read at 405 nm to

649

monitor the generation of pNP. The activity was expressed as nmol of pNP/min/mg

650

protein. 29



651

Figure 3. Biochemical properties of CYP9A61. (a) The activity of CYP9A61

652

expressed in Escherichia coli (E-CYP9A61) ( □ ) and Pichia pastoris

653

(P-CYP9A61) (△) at different pH; (b) The activity of E-CYP9A61 (□) and

654

P-CYP9A61 ( △ ) at different temperatures. (c) The thermostability of

655

E-CYP9A61 (□) and P-CYP9A61 (△). The error bars represent the standard

656

deviation (SD) of the mean of three replicates.

30


Page 30 of 36

Page 31 of 36


Table 1. Primer used in this study. Primer name

Primer sequence (5’-3’)

F1 R1

GGGGTACCATGTATCGTAACTACACCACG ACTAGCGGCCGCTCAATGATGATGATGATGATGATTCTTGCGTGGCCTAAAC GACTGGTTCCAATTGACAAGC GCAGCAATGCTGGCAATAGTA GCAAATGGCATTCTGACATCC

5′AOX-F α-factor R 3′AOX-R

The specific primers F1 and R1 complementary to the flanking sequences of the open reading frame (ORF) with Kpn Ⅰ and Not Ⅰ restriction enzyme sites (underlined) respectively. The 6×His tag sequences are in bold.



Page 32 of 36

Table 2. IC50 values for pyrethroids and inhibitor. IC50 (µM) Insecticide

Structure E-CYP9A61

P-CYP9A61

Cypermethrin

27.8±2.5

15.8±1.2

Permethrin

78.9±8.1

41.2±4.3

Lambda-cyhalothrin

19.7±1.5

10.3±0.9

Quercetin

0.4±0.08

0.2±0.015


Page 33 of 36


Figure 1 61x37mm (300 x 300 DPI)



Figure 2 70x99mm (300 x 300 DPI)


Page 34 of 36

Page 35 of 36


Figure 3 121x245mm (300 x 300 DPI)



TOC 36x17mm (300 x 300 DPI)


Page 36 of 36

Comparative Analysis of Recombinant Cytochrome P450 CYP9A61

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