Structural and functional characterization of OleP, a cytochrome P450 epoxidase

The identification of the molecular features critical for substrate binding and the determination of the molecular mechanisms through which it is processed represents fundamental aims in the study of a particular enzyme. Hence, the knowledge of its three-dimensional structure constitutes a starting point and a key tool for defining its properties. Previous studies concerning the cytochrome P450 OleP demonstrated its implication in the biosynthetic pathway that leads to the antibiotic oleandomycin biosynthesis in S. antibioticus. Whereas many enzymes involved in biosynthetic pathways show a high specificity toward their substrates, OleP was demonstrated to have activity on two metabolic intermediates in the pathway that leads to oleandomycin biosynthesis. Moreover, the reaction that OleP itself catalyzes is peculiar in the context of mono-oxygenation reactions promoted by P450 enzymes, considering that the epoxide moiety is introduced on two inactivated carbons (single C-C bond). Such a reaction is indeed not directly achievable in organic chemistry synthesis and unique in the bacterial P450s scenario. Accordingly, the natural versatility this enzyme was demonstrated to possess together with the opportunity to control and amplify it by means of enzymatic engineering, make this P450 an attractive candidate to be exploited as a tool for organic chemistry, novel antibiotics development or bioremediation purposes. Therefore, the aim of this thesis was the structural and functional characterization of OleP in the context of oleandomycin biosynthesis. Furthermore, given the current tendency in using P450 inhibitors in the treatment of infections as well as cardiovascular diseases and cancer, another aim of this work was to obtain structures of OleP in complex with two largely diffused drugs, ketoconazole and clotrimazole, two azole derivatives of different size and shape. In fact, obtaining P450 structures whose specificities and, presumably, active sites structures are very different from each other may contribute to map out a range of active sites structures available for P450s and, thereby, expand the database for homology modelling efforts. Hence, the availability of these structures may provide a rational for the design of more potent and/or selective inhibitors against individual forms of P450s.