Surveying a Pseudomonas aeruginosa-derived oxidoreductase activity

Abstract

Background and Objectives: Flavoprotein monooxygenases (FPMOs) participate in various biological processes, includ- ing lignin degradation, natural product biosynthesis, and xenobiotic detoxification. This study aimed to investigate the heter- ologous 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 de- creased 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.