title: Functional characterization of glutathione S-transferases associated with insecticide resistance in Tetranychus urticae.
abstract: The two-spotted spider mite Tetranychus urticae is one of the most important agricultural pests world-wide. It is extremely polyphagous and develops resistance to acaricides. The overexpression of several glutathione S-transferases (GSTs) has been associated with insecticide resistance. Here, we functionally expressed and characterized three GSTs, two of the delta class (TuGSTd10, TuGSTd14) and one of the mu class (TuGSTm09), which had been previously associated with striking resistance phenotypes against abamectin and other acaricides/insecticides, by transcriptional studies. Functional analysis showed that all three GSTs were capable of catalyzing the conjugation of both 1-chloro-2,4 dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene(DCNB) to glutathione (GSH), as well as exhibiting GSH-dependent peroxidase activity toward Cumene hydroperoxide (CumOOH). The steady-state kinetics of the T. urticae GSTs for the GSH/CDNB conjugation reaction were determined and compared with other GSTs. The interaction of the three recombinant proteins with several acaricides and insecticides was also investigated. TuGSTd14 showed the highest affinity toward abamectin and a competitive type of inhibition, which suggests that the insecticide may bind to the H-site of the enzyme. The three-dimensional structure of the TuGSTd14 was predicted based on X-ray structures of delta class GSTs using molecular modeling. Structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of TuGSTd14.
authors.affiliation: Department of Biology, University of Crete, Heraklion 71409, Greece.
authors.forename: Nena
authors.initials: N
authors.lastname: Pavlidi
authors.affiliation: Department of Biology, University of Crete, Heraklion 71409, Greece.
authors.forename: Vasilis
authors.initials: V
authors.lastname: Tseliou
authors.affiliation: Department of Biology, University of Crete, Heraklion 71409, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Riga
authors.affiliation: BayerCropScience AG, RD-SMR Pest Control Biology, Alfred Nobel Str. 50, Monheim D-40789, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Nauen
authors.affiliation: Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, Amsterdam 1098 XH, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Van Leeuwen
authors.affiliation: Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 IeraOdos Street, Athens GR-11855, Greece.
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, Heraklion Crete GR-700 13, Greece; Laboratory of Pesticide Science, Department of Crop Science, Agricultural University of Athens, 75 IeraOdos Street, Athens GR-11855, Greece. Electronic address: vontas@imbb.forth.gr.
authors.forename: John
authors.initials: J
authors.lastname: Vontas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007305:Insecticide Resistance / Q000502:physiology*; D007306:Insecticides / Q000494:pharmacology; D007559:Ivermectin / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D040102:Tetranychidae / Q000187:drug effects / Q000201:enzymology*
chemical_list: D019476:Insect Proteins; D007306:Insecticides; D011994:Recombinant Proteins; C048324:abamectin; D007559:Ivermectin; D005982:Glutathione Transferase
keywords: Abamectin; Enzyme modeling; Glutathione S-transferases; Insecticide resistance; Tetranychus urticae