title: The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.
abstract: FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
authors.forename: Vojtěch
authors.initials: V
authors.lastname: Sedláček
authors.affiliation: Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Klumpler
authors.affiliation: Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Jaromír
authors.initials: J
authors.lastname: Marek
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
authors.forename: Igor
authors.initials: I
authors.lastname: Kučera
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D005415:Flavins; D005420:Flavoproteins; D009249:NADP; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases