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Studies of bacterial and insect cytochromes P450 in degradation of pesticides

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Thesis type
(Thesis) M.Sc.
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Cytochromes P450 is a group of heme-containing enzymes with diverse catalytic activity that can be used for the biodegradation of environmental chemicals. Cytochrome P450cam (CYP101A1) from the soil bacterium Pseudomonas putida is known for hydroxylating camphor. Here, I have investigated the dehalogenation ability of two P450cam mutants, ES6 (G120S) and ES7 (V247F/D297N/K314E), in comparison to the wild-type (WT) enzyme. Six hexachlorinated persistent organic pollutants (POP), namely endosulfan (ES), ES diol, ES lactone, ES ether, ES sulfate and heptachlor, were tested since they are similarly structured to the native substrate. The mutated enzymes were capable of converting the selected substrates to phenols and o-quinones, which were detected using a colorimetric assay with 4-aminoantipyrine (4-AAP). Kinetic studies and statistical analysis were carried out and it was found that both ES6 and ES7 are significantly more active than the WT, with the highest activity noticed against ES ether and heptachlor. The western honey bee, Apis mellifera, is a vital pollinator of the ecosystem, however, its being threatened by the ectoparasitic mite, Varroa destructor. This pest is becoming immune towards commercially available pesticides, thus, new control agents have been previously synthesized that showed miticidal effects. Fortunately, insect cytochromes P450 are known to be responsible for the metabolism of such xenobiotics. Here, I have tested the ability of three potent dialkoxybenzene compounds, namely 1-allyloxy-4-propoxybenzene (3c{3,6}), 1,4-dipropoxybenzene (3c{3,3}) and 1,4-diallyloxybenzene (3c{6,6}), to get degraded by honey bee cytochromes P450. The formation of the dealkylated products was detected in abdomen extracts using a colorimetric assay with 4-aminoantipyrine (4-AAP). Kinetic studies and statistical analysis showed a downregulation of detectable P450 activity in the treated vs. the untreated extracts. Gas chromatography-mass spectrometry (GC-MS) quantitative assays were carried out and three dealkylated products were found, hydroquinone (HQ), 1-hydroxy-4-propoxybenzene (2c{3}) and 1-hydroxy-4-allyloxybenzene (2c{6}).
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Thesis advisor: Plettner, Erika
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