FAD synthase, the last enzyme of the pathway converting riboflavin to FAD, exists in humans in different isoforms, with isoforms 1, 2 and 6 being characterized at the functional and molecular levels. Isoform 2, the cytosolic and most abundant FADS, consists of two domains: a PAPS reductase C-terminus domain (here named FADSy) responsible for FAD synthesis, and an N-terminus molybdopterin-binding resembling domain (MPTb - here named FADHy), whose FAD hydrolytic activity is hidden unless both Co2+ and chemical mercurial reagents are added to the enzyme. To investigate the hFADS2 hydrolytic function under conditions closer to the physiological context, the hydrolytic activity was further characterized. Co2+ induced FAD hydrolysis was strongly stimulated in the presence of K+, reaching a Vmax higher than that of FAD synthesis. The pH dependence together with the inhibition of the hydrolysis by NaF and KI allow excluding that the reaction occurs via a NUDIX type catalysis. The K0.5 for K+ or Co2+ was 7.2 or 0.035 mM, respectively. Other monovalent or divalent cations can partially substitute K+ or Co2+. Reduced glutathione stimulated whereas NADH inhibited the hydrolytic activity. The latter aspects correlate with an interconnection of the homeostasis of NAD and FAD.
