Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 3; pp. 565 - 576
• Main Authors: Chronopoulou, Evangelia G., Vlachakis, Dimitrios, Papageorgiou, Anastassios C., Ataya, Farid S., Labrou, Nikolaos E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: binding properties; binding sites; Binding Sites - genetics; Biosensing Techniques - methods; Biosensor; biosensors; Catalysis; catalytic activity; Catalytic Domain - genetics; Directed Molecular Evolution - methods; Endosulfan - analysis; Endosulfan - isolation & purification; Environmental Monitoring - methods; Environmental Pollutants - analysis; Environmental Pollutants - isolation & purification; Enzymes, Immobilized - genetics; Enzymes, Immobilized - metabolism; Glutathione - metabolism; Glutathione transferase; Glutathione Transferase - chemistry; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Glycine max - enzymology; Glycine max - genetics; hydrophobicity; mutagenesis; Mutagenesis, Site-Directed; Mutant Proteins - metabolism; mutants; pesticides; Pesticides - analysis; Pesticides - isolation & purification; Pesticides determination; Phaseolus - enzymology; Phaseolus - genetics; phenolsulfonphthalein; Protein engineering; Protein Engineering - methods; protons; Reproducibility of Results; screening; standard deviation; Structure-Activity Relationship; xenobiotics; α-Endosulfan; H-site; RDS; Protein engineering; PTMOS; GST; α-Endosulfan; ANNs; TEOS; PEG; Glutathione transferase; G-site; Biosensor; MD; Pesticides determination; PvGmGSTUG; GSH
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2018.12.004

In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H+] by the catalytic reaction, measured by sol–gel entrapped indicators. The assay response at 562 nm was linear in the range pH = 4–7. Linear calibration curves were obtained for α-endosulfan in the range of 0–30 μΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps. •A structure-based design approach was used for engineering a mutant GST.•The mutant Phe117Ile displayed higher sensitivity towards pesticides.•The mutant was explored for the development of a novel optical biosensor for α-endosulfan.•The method was applied in real water samples without sample preparation steps.