title: Purification and characterization of NADH oxidase from the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus.
abstract: The enzyme NADH oxidase (EC 1.6.99.3) has been isolated from the two thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized. In both organisms the enzyme oxidizes specifically beta-NADH in the presence of molecular oxygen and requires the presence of a flavin cofactor, showing a high specificity for FAD. A stoicheiometric amount of hydrogen peroxide to NADH is formed as the end product of the reaction, indicating that both enzymes are two-electron donors. The purified enzymes exhibit quite different molecular properties. S. acidocaldarius NADH oxidase is a monomeric protein with an estimated molecular mass of about 27 kDa, whereas S. solfataricus NADH oxidase is a dimeric protein with a molecular mass of 35 kDa per subunit; S. solfataricus NADH oxidase is purified as an FAD-containing protein, whereas S. acidocaldarius NADH oxidase does not contain a flavin molecule. Furthermore, a comparison of the N-terminal amino acid sequence shows no similarities either between the two proteins or to any other NADH oxidases. Both enzymes are essentially thermophilic. In the temperature range 20-80 degrees C, the energy of activation is almost the same for both activities, suggesting that similar energetic parameters are required. Also both oxidases display a great stability to heat. The half-life of heat inactivation is about 180 min at 90 degrees C for S. acidocaldarius NADH oxidase and 77 min at 98 degrees C for the S. solfataricus enzyme. The activity of the two enzymes is inhibited by urea and guanidine and are regulated very differently by several organic solvents.
authors.affiliation: Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Masullo
authors.forename: G
authors.initials: G
authors.lastname: Raimo
authors.forename: A
authors.initials: A
authors.lastname: Dello Russo
authors.forename: V
authors.initials: V
authors.lastname: Bocchini
authors.forename: J V
authors.initials: JV
authors.lastname: Bannister
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D012997:Solvents; D013053:Spectrophotometry; D016944:Sulfolobus / Q000201:enzymology*; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*; D014508:Urea
chemical_list: D006146:Guanidines; D009097:Multienzyme Complexes; D012997:Solvents; D014508:Urea; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019791:Guanidine