Purification and Properties of NADH Oxidase from Bacillus megaterium

• Published in: Journal of biochemistry (Tokyo) Vol. 98; no. 6; pp. 1433 - 1440
• Main Authors: SAEKI, Yukikazu, NOZAKI, Mitsuhiro, MATSUMOTO, Kunio
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1985
• Subjects: Adenosine Diphosphate Ribose - metabolism; Amino Acids - analysis; Anaerobiosis; Analytical, structural and metabolic biochemistry; Bacillus megaterium; Bacillus megaterium - enzymology; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biological and medical sciences; Enzymes and enzyme inhibitors; Flavin Mononucleotide - metabolism; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Molecular Conformation; Multienzyme Complexes - isolation & purification; Multienzyme Complexes - metabolism; NADH oxidase; NADH, NADPH Oxidoreductases - isolation & purification; NADH, NADPH Oxidoreductases - metabolism; NADP - metabolism; Oxidoreductases; Oxygen - metabolism; Spectrophotometry; Substrate Specificity; Purification; Bacillales; Affinity chromatography; Enzyme; Bacteria; Bacillaceae; Catalyst activity; Bacillus megaterium; Physicochemical properties
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a135411

NADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD+ and hydrogen peroxide was purified from Bacillus megaterium by 5′-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr=52,000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mM KP1 buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH:oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.