Masullo M, Raimo G, Dello Russo A, Bocchini V, Bannister J V
Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Italy.
Biotechnol Appl Biochem. 1996 Feb;23(1):47-54.
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.
已从嗜热嗜酸古菌嗜酸热硫化叶菌和嗜热栖热硫化叶菌中分离并鉴定出NADH氧化酶（EC 1.6.99.3）。在这两种生物体中，该酶在分子氧存在下特异性氧化β-NADH，并且需要黄素辅因子的存在，对FAD表现出高度特异性。反应的终产物是化学计量的过氧化氢与NADH，这表明这两种酶都是双电子供体。纯化后的酶表现出相当不同的分子特性。嗜酸热硫化叶菌NADH氧化酶是一种单体蛋白，估计分子量约为27 kDa，而嗜热栖热硫化叶菌NADH氧化酶是一种二聚体蛋白，每个亚基分子量为35 kDa；嗜热栖热硫化叶菌NADH氧化酶作为含FAD的蛋白被纯化，而嗜酸热硫化叶菌NADH氧化酶不含有黄素分子。此外，对N端氨基酸序列的比较表明，这两种蛋白之间以及与任何其他NADH氧化酶之间均无相似性。这两种酶本质上都是嗜热的。在20-80摄氏度范围内，两种酶活性的活化能几乎相同，这表明需要相似的能量参数。而且这两种氧化酶对热都表现出很高的稳定性。嗜酸热硫化叶菌NADH氧化酶在90摄氏度时热失活的半衰期约为180分钟，嗜热栖热硫化叶菌的酶在98摄氏度时为77分钟。这两种酶的活性受到尿素和胍的抑制，并且受到几种有机溶剂的调节方式非常不同。
