A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

J Bacteriol. 2000 Sep;182(18):5046-51. doi: 10.1128/JB.182.18.5046-5051.2000.

Abstract

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Anaerobiosis
  • Bacteria, Anaerobic / enzymology*
  • Bacteria, Anaerobic / genetics*
  • Cloning, Molecular
  • Disulfides / analysis
  • Escherichia coli Proteins
  • Flavin-Adenine Dinucleotide / metabolism
  • Hydrogen Peroxide / metabolism
  • Kinetics
  • Models, Chemical
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / genetics*
  • Multienzyme Complexes / metabolism*
  • NADH, NADPH Oxidoreductases / chemistry
  • NADH, NADPH Oxidoreductases / genetics*
  • NADH, NADPH Oxidoreductases / metabolism*
  • Peroxidases / metabolism*
  • Peroxiredoxins
  • Recombinant Proteins / metabolism
  • Salmonella enterica / enzymology
  • Superoxide Dismutase / metabolism

Substances

  • Disulfides
  • Escherichia coli Proteins
  • Multienzyme Complexes
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide
  • Hydrogen Peroxide
  • Peroxidases
  • Peroxiredoxins
  • ahpC protein, E coli
  • Superoxide Dismutase
  • NADH oxidase
  • NADH, NADPH Oxidoreductases

Associated data

  • GENBANK/AB018435