Abstract
The genome of Drosophila melanogaster encodes a diverse array of cytochrome P450 enzymes, several of which are responsive to xenobiotic substances such as pesticides and terpenoid phytochemicals. This study investigated the transcriptional regulation and functional role of the Cyp6a2 gene in response to dichlorodiphenyltrichloroethane (DDT) and (-)-α-pinene exposure. In wild-type flies, Cyp6a2 expression was markedly upregulated following DDT and (-)-α-pinene treatment, respectively. To investigate the role of Cyp6a2 in detoxification, we generated a GAL4-dependent Cyp6a2 RNAi knockdown line using the Da-GAL4 driver. These knockdown flies exhibited significantly lower survival rates upon exposure to both compounds, indicating a protective function for the CYP6A2 enzyme. Moreover, immunodetection of active-Death caspase-1 (active-Dcp-1) in the midgut of DDT-treated knockdown flies further supported this protective role. For biochemical characterization, Cyp6a2 gene was cloned into a pCW vector, heterologously expressed in Escherichia coli, and the resulting recombinant protein was purified. Spectral analysis revealed that the ferric form of CYP6A2 enzyme predominantly existed in a low-spin state. Ligand-binding titrations demonstrated characteristic type I spectral shifts for both DDT and (-)-α-pinene, with strong binding affinities (Kd = 15.8 ± 3.4 μM and 18.8 ± 1.3 μM, respectively). Molecular docking suggested that both ligands bind near the heme group within the active site of P450 enzyme, oriented for oxidation. These results enhance our understanding of the physiological and biochemical functions of CYP6A2 in protecting D. melanogaster from xenobiotic stress caused by pesticides and phytochemicals.
