Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus.
Three NADH oxidase encoding genes noxA-1, noxB-1 and noxC were cloned from the genome of Archaeoglobus fulgidus, expressed in Escherichia coli, and the gene products were purified and characterized. Expression of noxA-1 and noxB-1 resulted in active gene products of the expected size. The noxC gene was expressed as well but the protein produced showed no activity in the standard Nox assay. NoxA-1 and NoxB-1 are both FAD-containing enzymes with subunit molecular masses of 48 and 69 kDa, respectively. NoxA-1 exists predominantly as homodimer, NoxB-1 as monomer. NoxA-1 and NoxB-1 showed pH optimum of 8.0 and 6.5, with specific NADH oxidase activities of 5.8 U.mg-1 and 4.1 U.mg-1, respectively. Both enzymes were specific for NADH as electron donor, but with different apparent Km values (NoxA-1, 0.13 mm; NoxB-1, 0.011 mm). The apparent Km values for oxygen differed significantly (NoxA-1, 0.06 mm; NoxB-1, 2.9 mm). In contrast with all mesophilic homologues, both enzymes were found to produce predominantly H2O2 instead of H2O. Despite apparent similarities, NoxB-1 is essentially different from NoxA-1. Whereas NoxA-1 resembles typical H2O-producing Nox enzymes that are expected to have a role in oxidative stress defence, NoxB-1 belongs to a small group of enzymes that is involved in catalysing the reduction of unsaturated acids and aldehydes, suggesting a role in fatty acid oxidation. Moreover, NoxB-1 contains a ferredoxin-like motif, which is absent in NoxA-1.
Kengen
Servé W M
SW
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, The Netherlands. serve.kengen@wur.nl
van der Oost
John
J
de Vos
Willem M
WM
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010802:Phylogeny; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D019843:Archaeal Proteins; D009097:Multienzyme Complexes; D016877:Oxidants; D020033:Protein Isoforms; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D010100:Oxygen