The role of cysteine residues in human FAD synthase (isoform 2)

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into FAD. At the moment two isoforms of the human FADS have been characterized, being involved in FAD synthesis in mitochondria and cytosol (1). hFADS is organized in two domains: the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase domain, which is the catalytic domain, and the resembling molybdopterin-binding domain (MPTbd), which performs a kinetic regulatory role (2). Using C. glabrata FMNAT as a template, we built the homology model of the PAPS reductase domain. Mass spectrometry and DTNB titration experiments allowed us to reveal the presence of four disulfide bridges in hFADS2, three of them located in close proximity to the PAPS reductase domain, and to validate the proposed model. In the frame of the hypothesis that hFADS is a component of a machinery that delivers FAD to cognate apo-flavoproteins in a tightly controlled process, the redox residues of cysteines require particular attention (2, 3). Redox changes of hFADS are ascertained here by the shift of the bands observed in non-reducing vs reducing conditions on SDS-PAGE. The involvement of cysteines in hFADS catalytic cycle is demonstrated by the strong inhibitor effects of thiol reagents on FAD synthesis rate. To verify the structure/function relationships of these redox residues we performed single substitutions of cysteines with alanine by site directed mutagenesis using overlap extension procedure starting from the most conserved residues, and from those closest to the active site in the homology model. All the purified mutants are able to synthesize FAD, C303A being the most significantly impaired. (1)Torchetti, E.M. et al. Mitochondrion-2010, 10, 263–273 (2)Miccolis A. et al. Int. J. Mol. Sci.-2012, 13, 16880-16898 (3)Torchetti, E.M et al. FEBS J.-2011, 278, 4434–4449 Acknowledgments: This work was supported by grants from PON-ricerca e competitivita 2007-2013 (PON project 01_00937) to M.B.