title: Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins.
abstract: A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello (DBBM), Università degli Studi di Bari, Bari, Italy.
authors.forename: Enza M
authors.initials: EM
authors.lastname: Torchetti
authors.forename: Francesco
authors.initials: F
authors.lastname: Bonomi
authors.forename: Michele
authors.initials: M
authors.lastname: Galluccio
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Gianazza
authors.forename: Teresa A
authors.initials: TA
authors.lastname: Giancaspero
authors.forename: Stefania
authors.initials: S
authors.lastname: Iametti
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
authors.forename: Maria
authors.initials: M
authors.lastname: Barile
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D044943:Fatty Acid Desaturases; C533241:FADS2 protein, human