College of Plant Sciences, Jilin University, Changchun 130-062, China.
Protein J. 2010 Nov;29(8):609-16. doi: 10.1007/s10930-010-9284-y.
NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.
NADH 氧化酶（NOXs）是解毒氧化应激和再生氧化吡啶核苷酸的重要酶。在本研究中，从 Thermococcus kodakarensis KOD1（NOXtk）中重组表达了一种 NOX，并将其纯化至均一性。NOXtk 表现出 NADH 氧化酶活性，该活性可被氧化抑制。在生理条件下，未氧化和氧化的 NOXtk 分别形成二聚体和六聚体。将单个半胱氨酸残基 Cys45 突变为丙氨酸（NOXtkC45A）会降低 NADH 氧化酶活性，但不影响二聚体或六聚体的形成，表明寡聚化不是通过二硫键形成发生的。下拉测定结果表明，来自 Thermococcus kodakarensis KOD1 的一种 ATP/NAD 激酶（ANKtk）与 NOXtk 结合。使用几种测定方法表明，ANKtk 只能与氧化的六聚体 NOXtk 结合，通过这种结合抑制 ANKtk 活性。因为 ANKtk 将 NADH 转化为 NADPH（氧化应激保护的重要因素），因此提出了一个基于体外结果的模型，即在有氧条件下 NOXtk 的六聚化会抑制 NOXtk 和 ANKtk 的活性，从而使细胞对氧化应激诱导的死亡敏感。
