Enzymes: NADH oxidases (EC 184.108.40.206); NADH peroxidase (EC 220.127.116.11); disulfide reductase (18.104.22.168).
Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus
Article first published online: 9 JUN 2003
European Journal of Biochemistry
Volume 270, Issue 13, pages 2885–2894, July 2003
How to Cite
Kengen, S. W. M., van der Oost, J. and Vos, W. M. d. (2003), Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus. European Journal of Biochemistry, 270: 2885–2894. doi: 10.1046/j.1432-1033.2003.03668.x
- Issue published online: 12 JUN 2003
- Article first published online: 9 JUN 2003
- (Received 28 February 2003, revised 25 April 2003, accepted 14 May 2003)
- NADH oxidase;
- oxygen stress
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.