tailieunhanh - Báo cáo khoa học: Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus

Three NADH oxidase encoding genesnoxA-1, noxB-1 andnoxCwere cloned from the genome ofArchaeoglobus fulgidus, expressed inEscherichia coli, and the gene products were purified and characterized. Expression ofnoxA-1and noxB-1resulted in active gene products of the expected size. ThenoxCgene was expressed as well but the protein pro-duced showed no activity in the standardNox assay. NoxA-1 and NoxB-1 are both FAD-containing enzymes with subunit molecular masses of 48 and 69 kDa, respectively. | Eur. J. Biochem. 270 2885-2894 2003 FEBS 2003 doi Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus Serve W. M. Kengen John van der Oost and Willem M. de Vos Laboratory of Microbiology Department of Agrotechnology and Food Sciences Wageningen University the Netherlands 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 and with specific NADH oxidase activities of U-mg-1 and U-mg-1 respectively. Both enzymes were specific for NADH as electron donor but with different apparent Km values NoxA-1 mM NoxB-1 mM . The apparent Km values for oxygen differed significantly NoxA-1 mM NoxB-1 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. Keywords Archaeoglobus flavoprotein NADH oxidase oxygen stress. Archaeoglobus fulgidus is a strictly anaerobic hyperthermo-philic archaeon that has been isolated from marine hydrothermal environments as .