title: Engineering sensitive glutathione transferase for the detection of xenobiotics.
abstract: Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20 microM. The method's reproducibility was in the order of +/-3-5% and malathion recoveries were 96.7+/-2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme.
authors.affiliation: Department of Agricultural Biotechnology, Laboratory of Enzyme Technology, Agricultural University of Athens, Athens, Greece.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Kapoli
authors.forename: Irene A
authors.initials: IA
authors.lastname: Axarli
authors.forename: Dimitris
authors.initials: D
authors.lastname: Platis
authors.forename: Maria
authors.initials: M
authors.lastname: Fragoulaki
authors.forename: Mark
authors.initials: M
authors.lastname: Paine
authors.forename: Janet
authors.initials: J
authors.lastname: Hemingway
authors.forename: John
authors.initials: J
authors.lastname: Vontas
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008294:Malathion / Q000032:analysis*; D009154:Mutation; D011199:Potentiometry; D015202:Protein Engineering; D015262:Xenobiotics / Q000032:analysis*; D003313:Zea mays / Q000201:enzymology
chemical_list: D004800:Enzymes, Immobilized; D015262:Xenobiotics; D005982:Glutathione Transferase; D008294:Malathion