RT Journal Article SR Electronic(1) A1 Case, Christopher L. A1 Rodriguez, Jason R. A1 Mukhopadhyay, BiswarupYR 2009 T1 Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii JF Microbiology, VO 155 IS 1 SP 69 OP 79 DO https://doi.org/10.1099/mic.0.024265-0 PB Microbiology Society, SN 1465-2080, AB Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit)−1, showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit)−1 and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were ≥10 and ≥95 °C, respectively. At pH 7 and 83 °C, apparent K m values for NADH and O2 were 3 μM and 1.9 mM, respectively, and the specific activity at 1.4 mM O2 was 60 μmol min−1 mg−1; 62 % of NADH-derived reducing equivalents were recovered as H2O2 and the rest probably generated H2O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5′-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high K m, O2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.024265-0