title: Bacterial over-expression and purification of the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain of human FAD synthase: functional characterization and homology modeling.
abstract: FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3'phosphoadenosine 5'phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble "truncated" form of hFADS lacking the N-terminal domain (Δ(1-328)-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ(1-328)-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ(1-328)-hFADS enzyme shows a slight, but not significant, change of K(m) values (0.24 and 6.23 µM for FMN and ATP, respectively) and of k(cat) (4.2 × 10-2 s-1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ(1-328)-hFADS versus wild-type protein.
authors: Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari "A. Moro", via Orabona 4, I-70126, Bari, Italy. maria.barile@uniba.it.
authors: Angelica
authors: A
authors: Miccolis
authors: Michele
authors: M
authors: Galluccio
authors: Teresa Anna
authors: TA
authors: Giancaspero
authors: Cesare
authors: C
authors: Indiveri
authors: Maria
authors: M
authors: Barile
mesh_terms: D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase