Structural characterization of the human enzyme FAD synthase

FMN and FAD are redox cofactors of a wide class of enzymes, known as flavoproteins. It is important to understand their synthesis pathway which, in eukaryotes and prokaryotes, is catalysed by two enzymes, riboflavin kinase (RFK) and FAD synthase (FADS). FADS is the second enzyme in the FAD forming pathway and adenylates FMN into FAD. In humans, this protein is present in different isoforms (the most represented are hFADS1 and hFADS2) and organized in two domains: at the C-terminal of the protein, a PAPS reductase domain catalyses the synthesis of FAD, while its N-terminal (MPTb) domain displays pyrophosphatase activity toward adenine dinucleotides, including FAD. To elucidate the structure of this enzyme, 3D structures of the protein have been obtained by homology modelling, but no experimental structure is available to date. This research project, performed in collaboration with the University of Bari and the University of Calabria, is part of this context and concerns the structural characterization of the recombinant full-length hFADS2, as well as the characterization of the crystal structure of the isolated PAPS-reductase domain, in the apo form and in complex with FAD, obtained by X-Ray diffraction. Different solutions and precipitants were used to set the crystallization conditions, then diffraction experiments have been performed at the European synchrotron Radiation Facility (ESRF) in Grenoble. Lastly, the functional characterization of the isolated N-terminal domain, whose function remained unclear for years, was performed by using Isothermal titration calorimetry (ITC). A side project of this thesis concerns the characterization of Electron Transfer Flavoprotein (ETF) variants associated with Multiple Acyl-CoA Dehydrogenase Deficiency (MADD). MADD is a rare genetic disease from the group of inborn error of metabolism (IEM), mainly determined by defects on ETF and ETF:QO proteins. This project has been carried out in collaboration with the Protein Misfolding and Amyloids in Biomedicine Laboratory of the University of Lisbon. The following ETF variants have been expressed in E. coli: ETFα:p.Leu77Val, ETFα:p.Arg104Lys, ETFα:p.Arg231Cys, ETFα:p.Gly237Val and ETFα:p.Gly249Arg. ETFα:p.Gly249Arg, which presented the highest expression yields as soluble protein, was subjected to structural, conformational and functional characterization of the protein was performed by circular dichroism (CD), fluorescence spectroscopy, differential scanning fluorimetry (DSF) and compared with the wild-type protein. Finally, the investigation of the mutant’s conformational stability was carried out by thermal denaturation.