title: Role of cysteine 337 and cysteine 340 in flavoprotein that functions as NADH oxidase from Amphibacillus xylanus studied by site-directed mutagenesis.
abstract: A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues. The complete reduction of enzyme by dithionite requires 6 electrons. Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides. In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant). Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state. These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. The catalytic activity of the three enzymes was examined by steady-state analysis. The Km for NADH and oxygen and the kcat value of these mutant enzymes were essentially the same as those of wild type. The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme. The NADH:5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme. The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH2. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH2 can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.
authors.affiliation: Department of Agricultural Chemistry, Tokyo University of Agriculture, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ohnishi
authors.forename: Y
authors.initials: Y
authors.lastname: Niimura
authors.forename: M
authors.initials: M
authors.lastname: Hidaka
authors.forename: H
authors.initials: H
authors.lastname: Masaki
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: T
authors.initials: T
authors.lastname: Uozumi
authors.forename: T
authors.initials: T
authors.lastname: Nishino
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D005420:Flavoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006092:Gram-Negative Facultatively Anaerobic Rods / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry
chemical_list: D005420:Flavoproteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine