Development of new bioactive molecules through cytochromes P450-mediated biocatalysis

Cytochromes P450s are large family of heme-thiolate proteins involved in a wide variety of biochemical reactions. Their versatility in catalysed reactions accepting diverse substrates, is fundamental in production of secondary metabolites and xenobiotics metabolism. Particularly noteworthy are self-sufficient P450s, which do not require an external redox partner to transfer electrons from NADPH. An example of such enzymes is P450 BM3 where reductase domain is fused directly to the catalytic domain, forming a multi-domain structure within a single polypeptide chain. The flexibility of the enzyme underpins their significant biotechnological potential as biocatalysts, evidenced by the production of numerous high-value compounds utilized in fields like pharmaceuticals and drug-discovery. In this thesis, the P450 BM3 A2 mutant was used to produce novel metabolites of natural compounds, to improve their biological activity. The compound used as a substrate was bergamottin, one of the most abundant furanocoumarin present in various citrus plants, having relevant bioactive features. The work was done in three parts: in silico analysis of interactions between protein and substrate, in vitro metabolism, and biological activity tests. First, molecular docking of bergamottin in the active site of BM3 A2 was performed using the YASARA software. The that there was a possible interaction of the substrate and the active site of the mutant. In the second part, an indirect method, the consumption of NADPH used as a cofactor in the reductive oxidation of substrate, allowed confirming the ongoing reaction. Afterwards, the HPLC was utilized in order to separate and isolate reaction products. Three compounds arising from bergamottin oxidation were identified. These were separated, collected, and concentrated for biological activity tests. The final step in the research involved the biological assays that would assess the antioxidant and antimicrobial potencies of the oxidized compounds. In this respect, antioxidant activity was measured using the ABTS assay. Results of this assay suggests that there is no significant difference in radical-scavenging capacities between bergamottin and its metabolites. In parallel with ABTS assay was also tested the antimicrobial activity. In this test proliferation of E. coli, a gram-negative bacteria, was measured when incubated with bergamottin or its metabolites. Two different methods were used, and their usefulness was compared. The first one, the serial dilution method with incubation on agar plates was tested but due to the high variability of results, it was not suitable for statistical evaluation. The second method was the microdilution method with cell growth in liquid medium on 96-well plate. What emerged from our study was that the 2 out of the 3 metabolites of bergamottin, precisely 2’,3’,6’,7’-diepoxybergamottin and 2’,3’ -epoxybergamottin are able to inhibit the growth of E. coli DH5α. This effect was not noted for third bergamottin metabolite 6’,7’-epoxybergamottin, and for bergamottin itself. These findings indicate that the P450 BM3 A2 can be used as a biocatalyst to introduce small changes in the structure of plant secondary metabolites, and that these small modifications can alter their biological properties.