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Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pest Helicoverpa armigera

Published online by Cambridge University Press:  05 June 2017

D. Liu ,
K. Tian ,
Y. Yuan ,
M. Li ,
M. Zheng  and
X. Qiu
D. Liu
Affiliation:
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 10049, China
K. Tian
Affiliation:
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 10049, China
Y. Yuan
Affiliation:
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
M. Li
Affiliation:
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
M. Zheng
Affiliation:
College of Science, China Agricultural University, Beijing 100193, China
X. Qiu*
Affiliation:
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
*
*Author for correspondence: Phone: +86-10-64807231 Fax: +86-10-64807099 E-mail: [email protected]
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Abstract

Cytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12 and CYP9A17) from the polyphagous pest Helicoverpa armigera in the facile Escherichia coli. The functionality of E. coli produced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed in E. coli with a content of about 1.0–1.5 nmol mg−1 protein in membrane preparations. In vitro assays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatest kcat/Km value for MROD, and CYP9A17 for EROD, while the lowest kcat/Km values for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.

Keywords

Helicoverpa armigeracytochrome P450CYP9Afunctional expressionalkoxyresorufin O-dealkylaseEscherichia coli
Type
Research Papers
Information
Bulletin of Entomological Research , Volume 108 , Issue 1 , February 2018 , pp. 77 - 83
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Copyright
Copyright © Cambridge University Press 2017 

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Footnotes

†

These authors contributed equally to this work.

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