Biocatalisi mediata da citocromi P450 per la produzione di agenti farmacologici: l'importanza dei composti naturali,

CYP102A1, known also as P450 BM3 WT, was the third cytochrome P450 discovered in Bacillus megaterium. It is the most studied bacterial P450 due to its broad substrate specificity and catalytic efficiency that is achieved thanks to its organization: the P450 heme domain (BMP), that contains the active site, is fused to the reductase domain (BMR), where the cofactors FAD and FMN allow the electron transfer necessary for the catalysis from the donor NADPH to the BMP. It is involved into the fatty acid metabolism as sub-terminal hydroxylase, although other substrates and reactions have been reported making this enzyme an attractive target for different biocatalytic applications, such as late stage functionalization of bioactive molecules. Natural compounds are becoming more and more important, as many of them possess different biological activities of therapeutic interest and offer a variety of molecular scaffolds that can be used for further modifications. However, such modifications are often difficult to achieve with standard chemical methods and enzymes offer a valid alternative. Thus, a pool of enzymes including the P450 BM3 WT and four mutants, named Y146, Y77, A2 and B35 was tested to modify four selected natural compounds: berberine, sanguinarine, curcumin and piperine. These compounds were chosen after a step of in silico docking analysis, involving the software AutoDock Vina, to predict their possible binding in the active site of the enzymes available. First, NADPH consumption tests were performed to state the possible reactivity of the enzymes in the presence of the 4 substrates. Then, HPLC analysis was performed to confirm the presence of metabolites and the only substrate giving a positive result was piperine. All the enzymes were able to turn over this substrate producing at least six metabolites with one being the most abundant. The mutant A2 resulted the most active with a Km of 55.18 ± 11.83 µM and a yield of substrate conversion of almost 60 %. Finally, the antimicrobial activity of the reaction mixture was tested against E. coli ATCC 8739 and S. aureus ATCC 6538, using the microdilution assay. The reaction mixture resulted to reduce S. aureus growth when compared to the control. Moreover, the main reaction product was extracted through HPLC and tested again, confirming an increased antimicrobial potency of the piperine-derived product. This work, among the others, highlights the importance of biocatalysis in the field of pharmaceutical production for the late-stage functionalization of natural compounds.