Abstract
Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation takes place through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agricultural pest of plants within Solanaceae, have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 Å by X-ray crystallography. The signature motif VSDGPPSL was identified in the “G-site”, and it contains the catalytically active residue Ser14. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2, 4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB was inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.
Highlights
Glutathione S-transferases (GSTs) constitute a large superfamily of multifunctional enzymes that are ubiquitously present in both prokaryotes and eukaryotes [1,2,3,4]
The LdGSTu1 enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that the conjugation of GSH to CDNB could be inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in pesticide adaptation
The LdGSTu1 gene was cloned from the L. decemlineata susceptible and resistant strains and shared 100% sequence similarity with the gene XP_023027125.1 in National Center for Biotechnology Information (NCBI) database
Summary
Glutathione S-transferases (GSTs) constitute a large superfamily of multifunctional enzymes that are ubiquitously present in both prokaryotes and eukaryotes [1,2,3,4]. Insect cytosolic GSTs are divided into several classes based on their sequence similarities and structural properties: delta, epsilon, sigma, omega, zeta, theta, and unclassified classes [9,10,11]. We identified an unclassified GST gene, LdGSTu1 from the Colorado potato beetle (CPB, Leptinotarsa decemlineata [Say]) in Coleoptera, which represents the most species-rich eukaryotic order, containing about half of the described herbivorous insect species (>400,000) [23]. The mechanisms of insect adaptation to pesticides and plant allelochemicals involves many aspects, including decreased penetration [28], target site insensitivity [29,30], enhanced metabolic detoxification [31,32,33], increased excretion, sequestration, as well as behavioral resistance [30,34]. The LdGSTu1 enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that the conjugation of GSH to CDNB could be inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in pesticide adaptation
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