title: Significance of redox-active cysteines in human FAD synthase isoform 2.
abstract: FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126 Bari, Italy. Electronic address: angelica.miccolis@uniba.it.
authors.forename: Angelica
authors.initials: A
authors.lastname: Miccolis
authors.affiliation: Dipartimento di Biologia, Ecologia, Scienze della Terra, Unità di Biochimica e Biotecnologia Molecolare, Università della Calabria, via Bucci 4c, I-87036 Arcavacata di Rende, Italy. Electronic address: mgalluccio@unical.it.
authors.forename: Michele
authors.initials: M
authors.lastname: Galluccio
authors.affiliation: Dipartimento di Agraria, Università di Napoli "Federico II", Parco Gussone, Portici I-80055, Napoli, Italy. Electronic address: chiara.nitride@unina.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Nitride
authors.affiliation: Istituto di Biomembrane e Bioenergetica (IBBE), CNR, via Amendola 165/A, I-70126 Bari, Italy. Electronic address: teresaanna.giancaspero@uniba.it.
authors.forename: Teresa Anna
authors.initials: TA
authors.lastname: Giancaspero
authors.affiliation: Dipartimento di Agraria, Università di Napoli "Federico II", Parco Gussone, Portici I-80055, Napoli, Italy. Electronic address: ferranti@unina.it.
authors.forename: Pasquale
authors.initials: P
authors.lastname: Ferranti
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, via Celoria 2, I-20133 Milano, Italy. Electronic address: stefania.iametti@unimi.it.
authors.forename: Stefania
authors.initials: S
authors.lastname: Iametti
authors.affiliation: Dipartimento di Biologia, Ecologia, Scienze della Terra, Unità di Biochimica e Biotecnologia Molecolare, Università della Calabria, via Bucci 4c, I-87036 Arcavacata di Rende, Italy. Electronic address: indiveri@unical.it.
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, via Celoria 2, I-20133 Milano, Italy. Electronic address: francesco.bonomi@unimi.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bonomi
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari "A. Moro", via Orabona 4, I-70126 Bari, Italy; Istituto di Biomembrane e Bioenergetica (IBBE), CNR, via Amendola 165/A, I-70126 Bari, Italy. Electronic address: maria.barile@uniba.it.
authors.forename: Maria
authors.initials: M
authors.lastname: Barile
keywords: Disulfide bridge; FMN adenylyltransferase; Human FAD synthase; Mercury toxicity; Redox sensing; Thiol