FAD forming and destroying via human FAD synthase: a puzzle of modulated and modulating activities

FAD synthase (FADS or FMN:ATP adenylyl transferase) coded by human FLAD1 gene, is the last enzyme in the pathway converting riboflavin into the redox cofactor FAD.Mutations in FLAD1 gene are responsible for Riboflavin-Responsive and Non-Responsive MADD and Combined Respiratory-Chain Deficiency[1]. Alternative splicing of the FLAD1 gene generates different hFADS isoforms: the mitochondrial isoform 1 and the cytosolic isoform 2. These are bi-functional enzymes containing two domains: a PAPS domain at the C-terminus able to catalyze FAD synthesis (EC 2.7.7.2), a molybdopterin-binding domain at the N-terminus able to perform FAD hydrolysis (EC 3.6.1.18).We show here that theCo2+-dependent hydrolytic activity of hFADS2 is strongly stimulated in the presence of K+, reaching a Vmax even higher than that of FAD synthesis. hFADS2acts as a non-NuDiX hydrolaseand it could interconnect FAD and NAD homeostasis. Recently,in patients suffering for frameshift mutations in the FLAD1 gene, we revealed ashort transcript variant corresponding to isoform 6,containing the sole PAPS domain[1]. We overproduced and characterized this emergency protein, which is relevant for patient survival. It is able to synthesize, but not to hydrolyze FAD [2].In the aim to find a target for therapy intervention in patients harboring FADS defects, a variant of hFADS6 carrying the site-directed mutation D238A, expected to exhibit a higher Kcat, was also over-produced, purified and characterized.