Ricerc@Sapienza

StaN is a cytochrome P450 (P450) involved in the biosynthesis of the antibiotic and anticancer indolocarbazole staurosporine. It was identified in 2005 once the whole biosynthetic cluster of staurosporine in Streptomyces sp TP-A0274 was sequenced. It can be classified as a P450 given its high sequence homology with other members of this superfamily and the presence of several typical molecular signatures of P450s, e.g. the universally conserved cysteine residue as the thiolate ligand to the heme. Nevertheless, StaN is supposed to catalyze an uncommon reaction for a P450 because it involves the formation of the glycosidic C-N bond between the staurosporine aglycone and the deoxyaminosugar, allowing for the formation of the intermediate O-demethyl-N-demethyl-staurosporine. To date, StaN represents the very first example of a P450 involved in C-N bond formation and its mechanism of action is unknown. My goal is to determine the high-resolution tridimensional structure of StaN by using x-ray crystallography in order to identify the molecular features critical for its activity and to understand the molecular mechanism behind its unusual reaction. I will also carry on a functional characterization of this system by means of substrate and inhibitors binding experiments. This also implies the production of site-specific mutants directed on critical residues, that will be identified by the structure, in order to better understand their specific role in catalysis and with the further aim to manipulate and redirect StaN activity against alternative substrate molecules. The long-term aim of this project is to produce new indolocarbazole derivatives able to bypass the non-selectivity that until now has precluded the use of staurosporine in anticancer therapy.