Surveying a Pseudomonas aeruginosa-derived oxidoreductase activity.

Abstract

Background and objectives: Flavoprotein monooxygenases (FPMOs) participate in various biological processes, including lignin degradation, natural product biosynthesis, and xenobiotic detoxification. This study aimed to investigate the heterologous expression of FPMO and assess its functional activity by examining its capacity to inactivate ampicillin.

Materials and methods: In silico analyses were performed to predict the enzyme's secondary and tertiary structures. The target gene was isolated from Pseudomonas aeruginosa, cloned into the pET-22b vector, and expressed in Escherichia coli BL21 (DE3). Heterologous protein expression was examined using SDS-PAGE, and the protein was purified through nickel-affinity chromatography. Enzymatic activity was measured by spectrophotometric monitoring of NADPH oxidation at 340 nm. Antibacterial activity was tested using an agar well-diffusion assay, measuring the inhibition zone diameter in E. coli treated with ampicillin.

Results: Homology modeling indicated that the 3D structure of FPMO is very similar to cyclohexanone monooxygenase, supporting its classification as a Baeyer-Villiger monooxygenase (BVMO). Molecular docking proposed that ampicillin might be a substrate for FPMO, with predicted interactions at Ile141 and Val159. Enzymatic assays confirmed that FPMO catalyzes the oxidation of ampicillin, using FAD and NADPH as cofactors. Additionally, well-diffusion tests showed decreased ampicillin antibacterial activity after treatment. The enzyme's activity was further confirmed using an ampicillin inactivation assay.

Conclusion: The heterologously expressed protein was functionally active. These findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin.