Ueyama Takehiko, Sakuma Megumi, Ninoyu Yuzuru, Hamada Takeshi, Dupuy Corinne, Geiszt Miklós, Leto Thomas L, Saito Naoaki
From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan,
From the Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
J Biol Chem. 2015 Mar 6;290(10):6495-506. doi: 10.1074/jbc.M114.592717. Epub 2015 Jan 13.
NADPH oxidase (Nox) family proteins produce superoxide (O2 (⨪)) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2 (⨪) intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2 (⨪) dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2 (⨪) leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2 (⨪) even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2 (⨪) in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2 (⨪) leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2 (⨪). Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2 (⨪) release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2 (⨪) leakage. Although we identified Duox1 A-loop residues (His(1071), His(1072), and Gly(1074)) important for reducing O2 (⨪) release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2 (⨪)-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex.
NADPH氧化酶（Nox）家族蛋白通过将电子转移到分子氧上直接产生超氧化物（O₂⁻）。双氧化酶（Duoxes）也会产生O₂⁻中间体，尽管成熟的Duoxes分泌的最终产物是H₂O₂，这表明分子内O₂⁻歧化或其他机制有助于H₂O₂的释放。我们探索了影响Duox酶活性氧生成的结构决定因素。当与Duox激活因子1（DuoxA1）不匹配时，Duox2会出现O₂⁻泄漏。即使在正确匹配的组合中，包括Duox2 + DuoxA2和Duox2 + N端标记的DuoxA2，无论标签的类型或数量如何，Duox2都会释放O₂⁻。相反，Duox1在任何组合中都不会释放O₂⁻。具有Duox1的A环的嵌合Duox2没有O₂⁻泄漏；具有Duox2的A环的嵌合Duox1释放O₂⁻。此外，与DuoxA2共表达的具有Nox1或Nox5的A环的Duox2蛋白显示出增强的O₂⁻释放，与DuoxA1共表达的具有Nox1或Nox5的A环的Duox1蛋白出现了O₂⁻泄漏。尽管我们确定了对减少O₂⁻释放很重要的Duox1 A环残基（His（1071）、His（1072）和Gly（1074）），但将这些残基突变为Duox2的残基并不能将Duox1转化为释放O₂⁻的酶。通过免疫沉淀和内切糖苷酶H敏感性分析，我们发现Duoxes的A环与DuoxA的N端结合，形成更稳定、成熟的Duox-DuoxA复合物。总之，两种Duoxes的A环通过与相应激活因子的相互作用支持H₂O₂的产生，但Duox1 A环与DuoxA1之间的复合物形成导致酶复合物对H₂O₂释放的控制更严格。
