Bacterial Expression and Kinetic Analysis of Carboxylesterase 001D from Helicoverpa armigera.

Yongqiang Li,Chongling Cai,Xing Zhang,Mei Lu,Jianwei Liu,Zhiqing Ma,Yonghong Wang

doi:10.3390/ijms17040493

Abstract

Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup of Helicoverpa armigera esterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene 001D of H. armigera from China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D in Escherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with a Kcat between 0.35 and 2.29 s−1, and a Km between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towards β-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min−1·(μM−1·protein)). The enzyme was stable up to 40 °C and at pH 6.0–11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides in H. armigera.

Highlights

The cotton bollworm, Helicoverpa armigera, is a major crop pest in many parts of the world [1,2,3,4]
Thecarboxylesterase gene 001D was cloned from a cDNA library of the midgut of H. armigera from the susceptible Wuhan (WH) strain
The alignment showed that carboxylesterase 001D has highly conserved residues of a catalytic triad (S202-H443-E330) and a pentapeptide termed the nucleophilic elbow (G200-S201-S202-A203-G204) (Figure 1). 001D contains the other three subsites, including the leaving group pocket (M333-R334-I133), acyl pocket (F235-T287-F309), and

Summary

Introduction

The cotton bollworm, Helicoverpa armigera, is a major crop pest in many parts of the world [1,2,3,4]. Carboxylesterases (EC3.1.1.1) play a critical role in the detoxification of xenobiotic compounds and are implicated in pyrethroid and OP resistance in a wide variety of pest insect species [9,10,11,12,13,14]. Carboxylesterase-mediated metabolic resistance to OPs and pyrethroids can occur through two mechanisms. The first one involves mutations in the active site (oxyanion hole and acyl-binding pocket) of the enzyme that elevates the OP hydrolytic activity and reduces the activity towards carboxylesterase substrates, such as 1-naphthyl acetate [15,16,17,18]. The second mechanism involves overexpression of unaltered carboxylesterases based on gene amplification or up-regulated transcription, allowing for effective sequestration of the insecticides instead of hydrolysis [19,20,21,22,23]

Methods

Results

Conclusion