Characteristics of a water-forming NADH oxidase from Methanobrevibacter smithii, an archaeon in the human gut

NADH oxidases (NOXs) catalysing the oxidation of NADH to yield NAD(+) and H(2)O, H(2)O(2), or both play an important role in protecting organisms from oxidative stress and maintaining the balance of NAD(+)/NADH. A gene encoding NOX was identified from Methanobrevibacter smithii (NOX-ms), the predominant archaeon in the human gut ecosystem. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 48 kDa. NOX-ms was purified to homogeneity after expression in Escherichia coli. NOX-ms catalysed the oxidization of NADH and converted O(2) to H(2)O with an optimal pH of 7.5 and a temperature optimum of approximately 37°C. The V(max) and K(m) values were 42.6–44.1 unit/mg and 47.8–54.6 μM for NADH. The apparent V(max) and K(m) for oxygen were 189.5–196.1 unit/mg and 14.6–16.8 μM. The mutation analysis suggests that Cys(42) in NOX-ms plays a key role in the four-electron reduction of O(2) to H(2)O. Quantitative reverse transcription-PCR (RT-qPCR) revealed that transcription of NOX-ms was also up-regulated after exposing the cells to oxidative stress and glucose. Finally, the potential of NOX-ms as a target to control colonization of M. smithii and its possible applications are discussed.