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

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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