Construction and analysis of an enzyme system showing extremely high scavenging activity for peroxides

我们先前从有氧生长的缺乏呼吸链和过氧化氢酶的木Amphibacillus xylanus中纯化了一种具有过氧化物酶和氧化酶功能的酶系统（NADH oxidase- ahpc）。该酶体系对过氧化氢和烷基过氧化氢均具有很高的清除活性。为了建立一种有效消除反应过程中过量过氧化物的方法，对该应用进行了研究。该酶具有三个氧化还原中心，酶结合的FAD和两个二硫化物，FADH_2的电子依次通过一级反应的二硫化物（Cys^<337 b> -Cys^<340>）和二级反应的二硫化物（Cys~<128>-Cys^<131>）来还原AhpC的二硫化物。这些半胱氨酸的突变研究表明，在AhpC存在的情况下，不仅第一二硫，而且第二二硫也参与氢过氧化物还原酶的活性。从水热菌中纯化出一种耐高温的NADH氧化酶- ahpc体系，其氨基酸序列与木Amphibacillus xylanus酶体系的同源性约为70%，并在纯化过程中发现了一种复合物。用SDS-PAGE分析了木Amphibacillus xylanus中这些蛋白复合物对应的蛋白带。利用SDS-PAGE对木Amphibacillus xylanus的AhpC和NADH氧化酶混合物进行免疫印迹分析，结果显示，AhpC与NADH氧化酶和AhpC抗体均能形成蛋白条带，并在这些蛋白条带中观察到两种蛋白对应的n端氨基酸序列。在二硫还原剂β-巯基乙醇的存在下，野生NADH氧化酶对应的蛋白条带明显消失。缺乏游离硫醇Cys^<480>的突变酶没有显示出蛋白带，表明在NADH氧化酶中，Cys^<480>的游离硫醇在两个蛋白之间形成了稳定的交联。虽然在DLS分析和超离心分析中也观察到该络合物，但在凝胶过滤分析中没有检测到。因此，NADH氧化酶和AhpC形成的复合体对过氧化物酶活性很重要，但它们松散地结合在一起。少

We previously purified an enzyme system (NADH oxidase-AhpC) which functions as peroxidase and oxidase from aerobically grown Amphibacillus xylanus that lacks both respiratory chain and catalase. The enzyme system showed an extremely high scavenging activity for both hydrogen peroxide and alkyl hydroperoxide. In order to establish an effective elimination method of the excess peroxides in the reaction process, investigation is performed in this application.The enzyme has three redox centers, enzyme-bound FAD and two disulfides, and electrons from FADH_2 have been shown to pass sequentially through the primary reacting disulfide(Cys^<337>-Cys^<340>) and the second disulfide(Cys~<128>-Cys^<131>) to reduce the disulfide of AhpC. The mutation study of these cysteins indicated that not only the first disulfide but also the second disulfide participates in the hydroperoxide reductase activity exhibited in the presence of AhpC. A thermostable NADH oxidase-AhpC system whose amino acid sequences … More showed about 70% of identity to the enzyme system of Amphibacillus xylanus, has been purified from Thermus aquaticus, and a complex of these proteins was found in purification process. Protein bands corresponding the complex of these proteins of Amphibacillus xylanus were investigated by SDS-PAGE in the absence of the disulfide reducer.Immunoblot analysis of mixture of AhpC and NADH oxidase of Amphibacillus xylanus by SDS-PAGE revealed that formation of protein bands reacted with antibodies against both NADH oxidase and AhpC, and then N-terminal amino acid sequences corresponding to both proteins were observed in these protein bands. The protein bands corresponding to wild NADH oxidase were disappeared clearly in the presence of the disulfide reducer, β-mercapto ethanol. The mutant enzyme lacking the free thiol Cys^<480> showed no protein bands, indicating that in the NADH oxidase, the free thiolate of Cys^<480> forms a stable cross-link between the two proteins. Although the complex was also observed in DLS analysis and ultra cetrifugal analysis, that could not be detected in gel filtration analysis. Thus, the formed complex of the NADH oxidase and AhpC should be important for peroxidase activity but lossely bound together. Less