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 solfataricus. The enzyme is a homodimer with M(r) 38,000 per subunit (SsNOX38) containing 1 FAD molecule/subunit. It oxidizes NADH and, less efficiently, NADPH with the formation of hydrogen peroxide. The enzyme was resistant against chemical and physical denaturating agents. The temperature for its half-denaturation was 93 and 75 degrees C in the absence or presence, respectively, of 8 M urea. The enzyme did not show any reductase activity. The SsNOX38 encoding gene was cloned and sequenced. It accounted for a product of 36.5 kDa. The translated amino acid sequence was made of 332 residues containing two putative betaalphabeta-fold regions, typical of NAD- and FAD-binding proteins. The primary structure of SsNOX38 did not show any homology with the N-terminal amino acid sequence of a NADH oxidase previously isolated from S. solfataricus (SsNOX35) (Masullo, M., Raimo, G., Dello Russo, A., Bocchini, V. and Bannister, J. V. (1996) Biotechnol. Appl. Biochem. 23, 47-54). Conversely, it showed 40% sequence identity with a putative thioredoxin reductase from Bacillus subtilis, but it did not contain cysteines, which are essential for the activity of the reductase.
Arcari
P
P
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
Masullo
L
L
Masullo
M
M
Catanzano
F
F
Bocchini
V
V
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D014508:Urea
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D014508:Urea; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013880:Thioredoxin-Disulfide Reductase