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

我们之前从缺乏呼吸链和过氧化氢酶的好氧培养的木霉中纯化了一个具有过氧化物酶和氧化酶功能的酶系统(NADH氧化酶-AhpC)。该酶体系对过氧化氢和烷基过氧化氢均表现出极高的清除活性。为了建立一种有效的消除反应过程中过量过氧化氢的方法，在本应用中进行了研究。酶有三个氧化还原中心，酶结合的FAD和两个二硫键，FADH_2的电子被证明依次通过反应的第一个二硫键(Cys^&lt；337&gt；-Cys^&lt；340&gt；)和第二个二硫键(Cys~&lt；128&gt；-Cys^&lt；131&gt；)还原AHpC的二硫键。这些半胱氨酸的突变研究表明，在AhpC存在下，不仅第一个二硫键参与了过氧化氢还原酶的活性，第二个二硫键也参与了酶的活性。氨基酸序列为…的耐热NADH氧化酶-AHPC系统进一步的研究表明，从水生黄热菌中分离纯化的木霉蛋白与木霉的酶系统有约70%的同源性，并且在纯化过程中发现了这些蛋白的复合体。在没有二硫键还原剂的情况下，用SDS-PAGE分析了与这些蛋白的复合体相对应的蛋白条带；用SDS-PAGE对木霉AhpC和NADH氧化酶的混合物进行了免疫印迹分析，发现这些蛋白质带与抗NADH氧化酶和AhpC的抗体都有反应，并在这些蛋白质带中出现了与这两种蛋白相对应的N端氨基酸序列。在二硫键还原剂β-巯基乙醇存在下，与野生NADH氧化酶相对应的蛋白条带明显消失。缺失游离硫醇Cys^&lt；480&gt；的突变酶没有蛋白条带，说明在NADH氧化酶中，Cys^&lt；480&gt；的游离硫酸盐在两种蛋白质之间形成了稳定的交联键。虽然在DLS分析和超速离心分析中也观察到了该复合体，但在凝胶过滤分析中检测不到该复合体。因此，NADH氧化酶和AhpC形成的复合体对过氧化物酶活性是重要的，但结合在一起是有损失的。较少

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