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.
1. Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable and ever-present adversary. J Hosp Infect 2009; 73: 338-344.
2. World Health Organization (WHO) (2024). WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance [Internet]. Geneva: World Health Organization; [cited 2026 Jul 18]. Available from: https://www.who.int/publications/i/item/9789240093461
3. Kuddus MA, Paul AK. Global Dynamics of a Two-Strain Disease Model with Amplification, Nonlinear Incidence and Treatment. Iran J Sci 2023; 47: 259-274.
4. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019; 37: 177-192.
5. Salehibarmi M, Siroosi M. Effect of supplementation on biofilms and antibiotic efficacy against Pseudomonas aeruginosa. Iran J Microbiol 2025; 17: 861-874.
6. Majeed HT, Aljanaby AAJ. Antibiotic Susceptibility Patterns and Prevalence of Some Extended Spectrum Beta-Lactamases Genes in Gram-Negative Bacteria Isolated from Patients Infected with Urinary Tract Infections in Al-Najaf City, Iraq. Avicenna J Med Biotechnol 2019; 11: 192-201.
7. Markley JL, Wencewicz TA. Tetracycline-Inactivating Enzymes. Front Microbiol 2018; 9: 1058.
8. Liu LK, Abdelwahab H, Del Martin Campo JS, Mehra-Chaudhary R, Sobrado P, Tanner JJ. The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase. J Biol Chem 2016; 291: 21553-21562.
9. Li Y, Zhao L, Pu X, Geng B, Liu W, Gao J, et al. Flavin-dependent monooxygenases as versatile biocatalysts in biomanufacturing: mechanisms, engineering, and applications. Biotechnol Biofuels Bioprod 2026; 19: 42.
10. Kim DW, Thawng CN, Lee K, Wellington EMH, Cha CJ. A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria. Environ Int 2019; 127: 206-215.
11. Ceccoli RD, Bianchi DA, Rial DV. Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications. Front Microbiol 2014; 5: 25.
12. Gonzalo G de, Mihovilovic MD, Fraaije MW. Recent developments in the application of Baeyer-Villiger monooxygenases as biocatalysts. Chembiochem 2010; 11: 2208-2231.
13. Tolmie C, Smit MS, Opperman DJ. Native roles of Baeyer-Villiger monooxygenases in the microbial metabolism of natural compounds. Nat Prod Rep 2019; 36: 326-353.
14. Riebel A, Dudek HM, Gonzalo G de, Stepniak P, Rychlewski L, Fraaije MW. Expanding the set of rhodococcal Baeyer-Villiger monooxygenases by high-throughput cloning, expression and substrate screening. Appl Microbiol Biotechnol 2012; 95: 1479-1489.
15. Jacewicz A, Schnell R, Lindqvist Y, Schneider G. Crystal structure of the flavoenzyme PA4991 from Pseudomonas aeruginosa. Acta Crystallogr F Struct Biol Commun 2016; 72: 105-111.
16. Langendonk RF, Neill DR, Fothergill JL. The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies. Front Cell Infect Microbiol 2021; 11: 665759.
17. Green MR, Sambrook J (2012). Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.
18. Rose RL (2002). Measurements of flavin-containing monooxygenase (FMO) activities. In: Current Protocols in Toxicology. John Wiley & Sons, Chapter 4, Unit 4.9, pp. 4.9.1-4.9.11.
19. Orru R, Pazmiño DET, Fraaije MW, Mattevi A. Joint functions of protein residues and NADP(H) in oxygen activation by flavin-containing monooxygenase. J Biol Chem 2010; 285: 35021-35028.
20. Miri M, Yazdianpour S, Abolmaali S, Darvish Alipour Astaneh S. Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function. Avicenna J Med Biotechnol 2022; 14: 46-53.
21. van Berkel WJ, Kamerbeek NM, Fraaije MW. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. J Biotechnol 2006; 124: 670-689.
22. Schwarz-Linek U, Krödel A, Ludwig F-A, Schulze A, Rissom S, Kragl U, et al. Synthesis of Natural Product Precursors by Baeyer-Villiger Oxidation with Cyclohexanone Monooxygenase from Acinetobacter. Synthesis 2001; 2001: 947-951.
23. Kamerbeek NM, Olsthoorn AJJ, Fraaije MW, Janssen DB. Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase. Appl Environ Microbiol 2003; 69: 419-426.
24. Drees SL, Ernst S, Belviso BD, Jagmann N, Hennecke U, Fetzner S. PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa. J Biol Chem 2018; 293: 9345-9357.
25. Meng N, Li G, Zhang J, Zhu J, Kang X, Duan J, et al. A novel and evolutionarily distinct flavoprotein monooxygenase drives skatole degradation in Rhodococcus. Appl Environ Microbiol 2026; 92(4): e0022826.
26. Vera A, González-Montalbán N, Arís A, Villaverde A. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng 2007; 96: 1101-1106.
27. Li YJ, Zheng YC, Geng Q, Liu F, Zhang ZJ, Xu JH, et al. Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation. Bioresour Bioprocess 2021; 8: 81.
28. Fan X, Widodo WS, Rozeboom HJ, Fraaije MW. Engineered Escherichia coli Strains for Flavoprotein Research: From Production of Apoproteins to Incorporation of Flavin Derivatives. ACS Synth Biol 2026; 15: 2635-2648.
29. Rehdorf J, Zimmer CL, Bornscheuer UT. Cloning, expression, characterization, and biocatalytic investigation of the 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1. Appl Environ Microbiol 2009; 75: 3106-3114.
30. Fürst MJLJ, Gran-Scheuch A, Aalbers FS, Fraaije MW. Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts. ACS Catal 2019; 9: 11207-11241.
31. Minerdi D, Zgrablic I, Castrignanò S, Catucci G, Medana C, Terlizzi ME, et al. Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem. Antimicrob Agents Chemother 2016; 60: 64-74.
32. Gasparrini AJ, Markley JL, Kumar H, Wang B, Fang L, Irum S, et al. Tetracycline-inactivating enzymes from environmental, human commensal, and pathogenic bacteria cause broad-spectrum tetracycline resistance. Commun Biol 2020; 3: 241.
33. Catucci G, Zgrablic I, Lanciani F, Valetti F, Minerdi D, Ballou DP, et al. Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation. Biochim Biophys Acta 2016; 1864: 1177-1187.
34. Bailleul G, Yang G, Nicoll CR, Mattevi A, Fraaije MW, Mascotti ML. Evolution of enzyme functionality in the flavin-containing monooxygenases. Nat Commun 2023; 14: 1042.
2. World Health Organization (WHO) (2024). WHO bacterial priority pathogens list, 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance [Internet]. Geneva: World Health Organization; [cited 2026 Jul 18]. Available from: https://www.who.int/publications/i/item/9789240093461
3. Kuddus MA, Paul AK. Global Dynamics of a Two-Strain Disease Model with Amplification, Nonlinear Incidence and Treatment. Iran J Sci 2023; 47: 259-274.
4. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019; 37: 177-192.
5. Salehibarmi M, Siroosi M. Effect of supplementation on biofilms and antibiotic efficacy against Pseudomonas aeruginosa. Iran J Microbiol 2025; 17: 861-874.
6. Majeed HT, Aljanaby AAJ. Antibiotic Susceptibility Patterns and Prevalence of Some Extended Spectrum Beta-Lactamases Genes in Gram-Negative Bacteria Isolated from Patients Infected with Urinary Tract Infections in Al-Najaf City, Iraq. Avicenna J Med Biotechnol 2019; 11: 192-201.
7. Markley JL, Wencewicz TA. Tetracycline-Inactivating Enzymes. Front Microbiol 2018; 9: 1058.
8. Liu LK, Abdelwahab H, Del Martin Campo JS, Mehra-Chaudhary R, Sobrado P, Tanner JJ. The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase. J Biol Chem 2016; 291: 21553-21562.
9. Li Y, Zhao L, Pu X, Geng B, Liu W, Gao J, et al. Flavin-dependent monooxygenases as versatile biocatalysts in biomanufacturing: mechanisms, engineering, and applications. Biotechnol Biofuels Bioprod 2026; 19: 42.
10. Kim DW, Thawng CN, Lee K, Wellington EMH, Cha CJ. A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria. Environ Int 2019; 127: 206-215.
11. Ceccoli RD, Bianchi DA, Rial DV. Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications. Front Microbiol 2014; 5: 25.
12. Gonzalo G de, Mihovilovic MD, Fraaije MW. Recent developments in the application of Baeyer-Villiger monooxygenases as biocatalysts. Chembiochem 2010; 11: 2208-2231.
13. Tolmie C, Smit MS, Opperman DJ. Native roles of Baeyer-Villiger monooxygenases in the microbial metabolism of natural compounds. Nat Prod Rep 2019; 36: 326-353.
14. Riebel A, Dudek HM, Gonzalo G de, Stepniak P, Rychlewski L, Fraaije MW. Expanding the set of rhodococcal Baeyer-Villiger monooxygenases by high-throughput cloning, expression and substrate screening. Appl Microbiol Biotechnol 2012; 95: 1479-1489.
15. Jacewicz A, Schnell R, Lindqvist Y, Schneider G. Crystal structure of the flavoenzyme PA4991 from Pseudomonas aeruginosa. Acta Crystallogr F Struct Biol Commun 2016; 72: 105-111.
16. Langendonk RF, Neill DR, Fothergill JL. The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies. Front Cell Infect Microbiol 2021; 11: 665759.
17. Green MR, Sambrook J (2012). Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.
18. Rose RL (2002). Measurements of flavin-containing monooxygenase (FMO) activities. In: Current Protocols in Toxicology. John Wiley & Sons, Chapter 4, Unit 4.9, pp. 4.9.1-4.9.11.
19. Orru R, Pazmiño DET, Fraaije MW, Mattevi A. Joint functions of protein residues and NADP(H) in oxygen activation by flavin-containing monooxygenase. J Biol Chem 2010; 285: 35021-35028.
20. Miri M, Yazdianpour S, Abolmaali S, Darvish Alipour Astaneh S. Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function. Avicenna J Med Biotechnol 2022; 14: 46-53.
21. van Berkel WJ, Kamerbeek NM, Fraaije MW. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. J Biotechnol 2006; 124: 670-689.
22. Schwarz-Linek U, Krödel A, Ludwig F-A, Schulze A, Rissom S, Kragl U, et al. Synthesis of Natural Product Precursors by Baeyer-Villiger Oxidation with Cyclohexanone Monooxygenase from Acinetobacter. Synthesis 2001; 2001: 947-951.
23. Kamerbeek NM, Olsthoorn AJJ, Fraaije MW, Janssen DB. Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase. Appl Environ Microbiol 2003; 69: 419-426.
24. Drees SL, Ernst S, Belviso BD, Jagmann N, Hennecke U, Fetzner S. PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa. J Biol Chem 2018; 293: 9345-9357.
25. Meng N, Li G, Zhang J, Zhu J, Kang X, Duan J, et al. A novel and evolutionarily distinct flavoprotein monooxygenase drives skatole degradation in Rhodococcus. Appl Environ Microbiol 2026; 92(4): e0022826.
26. Vera A, González-Montalbán N, Arís A, Villaverde A. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng 2007; 96: 1101-1106.
27. Li YJ, Zheng YC, Geng Q, Liu F, Zhang ZJ, Xu JH, et al. Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation. Bioresour Bioprocess 2021; 8: 81.
28. Fan X, Widodo WS, Rozeboom HJ, Fraaije MW. Engineered Escherichia coli Strains for Flavoprotein Research: From Production of Apoproteins to Incorporation of Flavin Derivatives. ACS Synth Biol 2026; 15: 2635-2648.
29. Rehdorf J, Zimmer CL, Bornscheuer UT. Cloning, expression, characterization, and biocatalytic investigation of the 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1. Appl Environ Microbiol 2009; 75: 3106-3114.
30. Fürst MJLJ, Gran-Scheuch A, Aalbers FS, Fraaije MW. Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts. ACS Catal 2019; 9: 11207-11241.
31. Minerdi D, Zgrablic I, Castrignanò S, Catucci G, Medana C, Terlizzi ME, et al. Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem. Antimicrob Agents Chemother 2016; 60: 64-74.
32. Gasparrini AJ, Markley JL, Kumar H, Wang B, Fang L, Irum S, et al. Tetracycline-inactivating enzymes from environmental, human commensal, and pathogenic bacteria cause broad-spectrum tetracycline resistance. Commun Biol 2020; 3: 241.
33. Catucci G, Zgrablic I, Lanciani F, Valetti F, Minerdi D, Ballou DP, et al. Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation. Biochim Biophys Acta 2016; 1864: 1177-1187.
34. Bailleul G, Yang G, Nicoll CR, Mattevi A, Fraaije MW, Mascotti ML. Evolution of enzyme functionality in the flavin-containing monooxygenases. Nat Commun 2023; 14: 1042.
| Files | ||
| Issue | Vol 18 No 4 (2026) | |
| Section | Original Article(s) | |
| Keywords | ||
| Ampicillin Flavoprotein Oxidoreductase Pseudomonas aeruginosa | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Mohammadkhani M, Abolmaali S, Darvishalipour Astaneh S. Surveying a Pseudomonas aeruginosa-derived oxidoreductase activity. Iran J Microbiol. 2026;18(4):533-543.
