title: Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.
abstract: The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy.
authors.forename: Teresa A
authors.initials: TA
authors.lastname: Giancaspero
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy.
authors.forename: Matilde
authors.initials: M
authors.lastname: Colella
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Brizio
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy.
authors.forename: Graziana
authors.initials: G
authors.lastname: Difonzo
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy.
authors.forename: Giuseppina M
authors.initials: GM
authors.lastname: Fiorino
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Leone
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg Freiburg, Germany.
authors.forename: Roderich
authors.initials: R
authors.lastname: Brandsch
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bonomi
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano Milano, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Iametti
authors.affiliation: Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro Bari, Italy ; Dipartimento di Scienze della Vita, Istituto di Biomembrane e Bioenergetica, CNR Bari, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Barile
keywords: FAD; FAD synthase; dimethylglycine dehydrogenase; flavinylation; lysine specific demethylase 1; mitochondria; nucleus; nutri-epigenetics; rat