Park H J, Reiser C O, Kondruweit S, Erdmann H, Schmid R D, Sprinzl M
Laboratorium für Biochemie, Universität Bayreuth, Federal Republic of Germany.
Eur J Biochem. 1992 May 1;205(3):881-5. doi: 10.1111/j.1432-1033.1992.tb16853.x.
A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain. The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H2O2. The apparent Km values for NADH and NADPH are 4.14 microM and 14.0 microM (pH 7.2 at room temperature), respectively, with a sixfold greater kcat/Km values for NADH compared to NADPH. The enzyme uses O2 as an electron acceptor in the presence of either FAD, riboflavin 5'-phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2- as an intermediate. The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80 degrees C.
通过几步色谱法从嗜热栖热菌HB8中纯化出了一种NADH氧化酶。在变性条件下进行凝胶电泳并测定N端氨基酸序列后判断，纯化后的酶基本呈均一状态。它是一种含黄素腺嘌呤二核苷酸的单体黄素蛋白，表观分子量为25 kDa，FAD与多肽链的比例为1:1。纯化后的酶催化还原型NADH或NADPH的氧化反应并生成H2O2。在室温、pH 7.2条件下，NADH和NADPH的表观Km值分别为4.14 μM和14.0 μM，NADH的kcat/Km值是NADPH的6倍。该酶在FAD、核黄素5'-磷酸或核黄素作为辅因子存在的情况下，利用O2作为电子受体。此外，即使在没有黄素穿梭体的情况下，该酶也能够催化电子从NADH转移到各种其他电子受体（亚甲蓝、细胞色素c、对硝基蓝四唑、2,6-二氯靛酚和铁氰化钾）。使用这些人工电子受体时，未观察到超氧化物歧化酶对NADH氧化还原酶活性有明显抑制作用，这表明电子转移主要是从NADH直接到电子受体，而不是通过O2-作为中间体。纯化后的NADH氧化酶在pH 5.0时表现出最高活性，并且在高达80℃的高温下稳定。
