monoossigenasi contenenti flavina 3 umana: studi comparativi sulla proteina WT, sul mutante C397S e sulla variante polimorfica V257M

Human flavin-containing monooxygenase 3 (hFMO3) is a drug metabolizing enzyme involved in the detoxification process of drugs and xenobiotics that are mainly soft nucleophiles. The catalytic activity of FMO enzymes consists in the NADPH dependent N- or S-oxygenation of substrates leading to the generation of more polar and excretable compounds. Human FMO3, that is mainly expressed in the liver, bound to the membrane, is able to metabolize a wide range of substrates, such as Thrimethylamine, Methimazole, Ranitidine, Tamoxifen, Benzydamine and Sulindac Sulfide. Previous studies have reported that polymorphic variants of hFMO3 generally show a decreased enzyme activity and some of these are related to the development of thrimethylaminuria disease. In this study, WT hFMO3, C397S mutant and V257M polymorphic variant were expressed in E.coli, purified via ion exchange and nickel affinity chromatography and then characterized. The purified proteins were assayed for their activity towards Ethionamide (ETA, antitubercolar drug), Tozasertib and Danusertib (aurora kinase inhibitors). KM values for the wild type- and C397S mutant- catalyzed reactions are similar in the case of ETA, as proved by HPLC analyses of product formation, whereas V257M variant resulted having a 6 fold lower value of KM. HPLC-MS qualitative and quantitative analyses of Danusertib and Tozasertib oxygenation reactions demonstrated that WT protein has a higher substrate specificity, given by the kcat/KM ratio, for Danusertib than the polymorphic variant, while it was comparable for Tozasertib metabolism. A comparison of the stability of WT and the two variants was achieved through spectrophotometric measurements of the Tm: C397S resulted being less stable than V257M and the WT protein. However, the same assays performed in the presence of NADP+ showed that all the proteins are stabilized by the binding of NADP+ (3-9% increase in Tm value).