A NAD(P)H oxidase isolated from the archaeon Sulfolobus solfataricus is not homologous with another NADH oxidase present in the same microorganism - Biochemical characterization of the enzyme and cloning of the encoding gene

A NAD(P)H oxidase has been isolated from the archaeon Sulfolobus solfataric us. The enzyme is a homodimer with M-r 38,000 per subunit (SsNOX38) contain ing 1 FAD molecule/subunit, It oxidizes NADH and, less efficiently, NADPH w ith the formation of hydrogen peroxide. The enzyme was resistant against ch emical and physical denaturating agents, The temperature for its half-denat uration was 93 and 75 degrees C in the absence or presence, respectively, o f 8 hr urea, The enzyme did not show any reductase activity. The SsNOX38 en coding gene was cloned and sequenced. It accounted for a product of 36.5 kD a. The translated amino acid sequence was made of 332 residues containing t wo putative beta alpha beta-fold regions, typical of NAD- and FAD-binding p roteins. The primary structure of SsNOX38 did not show any homology with th e N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M,, Raimo, G,, Dello Russo, A, Bocchin i, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem, 23, 47-54), Co nversely, it showed 40% sequence identity with a putative thioredoxin reduc tase from Bacillus subtilis, but it did not contain cysteines, which are es sential for the activity of the reductase.