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

具有极高过氧化物清除活性的酶系统的构建和分析

基本信息

  • 批准号:
    14560078
  • 负责人:
  • 金额:
    $ 1.86万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    2002
  • 资助国家:
    日本
  • 起止时间:
    2002 至 2003
  • 项目状态:
    已结题

项目摘要

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
我们以前纯化的酶系统(NADH氧化酶-AhpC),其功能作为过氧化物酶和氧化酶从有氧生长的Amphibacillusxylanus,缺乏呼吸链和过氧化氢酶。该酶体系对过氧化氢和烷基过氧化氢均有极高的清除活性。为了建立一种有效消除反应过程中过量过氧化物的方法,本研究发现该酶具有三个氧化还原中心,即酶结合FAD和两个二硫键,FADH_2产生的电子依次通过第一个反应二硫键(Cys ~<337>-Cys ~-<340>)和第二个反应二硫键(Cys~<128>-Cys ~-<131>)还原AhpC的二硫键。这些半胱氨酸的突变研究表明,不仅第一个二硫键,而且第二个二硫键参与的氢过氧化物还原酶活性的存在下表现出的AhpC。一种热稳定的NADH氧化酶-AhpC系统,其氨基酸序列 ...更多信息 从水生栖热菌(Thermus aquaticus)中分离纯化了一个与木聚糖双芽孢杆菌(Amphibacillus xylanus)的酶系统同源性约为70%的蛋白质,并在纯化过程中发现了这两种蛋白质的复合物。在不加二硫键还原剂的条件下,用SDS-PAGE对木端芽孢杆菌的AhpC和NADH氧化酶的混合物进行免疫印迹分析,结果表明,这两种蛋白质的复合物均能与抗NADH氧化酶和AhpC的抗体发生反应,并在蛋白质的N端观察到相应的氨基酸序列。在二硫键还原剂β-巯基乙醇存在下,野生型NADH氧化酶的蛋白条带明显消失。缺乏游离巯基Cys^的突变酶<480>没有显示出蛋白质条带,这表明在NADH氧化酶中,Cys^的游离巯基在<480>两种蛋白质之间形成了稳定的交联。在DLS分析和超离心分析中也观察到了这种复合物,但在凝胶过滤分析中却没有检测到。因此,NADH氧化酶和AhpC形成的复合物对于过氧化物酶活性应该是重要的,但松散地结合在一起。少

项目成果

期刊论文数量(20)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Y.Niimura et al.: "The NADH oxidase component of alkylhydroperoxide reductase. Reaction mechanism and physiological role in microorganisms"Flavins and Flavoproteins 2002. 393-398 (2002)
Y.Niimura等:“烷基氢过氧化物还原酶的NADH氧化酶成分。微生物中的反应机制和生理作用”Flavins and Flavo Proteins 2002. 393-398 (2002)
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K.Takeda et al.: "Distribution of Prx-linked Hydroperoxide Reductase Activity among Microorganismus"Biosci.Biotechnol.Biochem. 68. 20-27 (2004)
K.Takeda 等人:“微生物中 Prx 连接的氢过氧化物还原酶活性的分布”Biosci.Biotechnol.Biochem。
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S.Kawasaki et al.: "Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum"Arch.Microbiol. in press.
S.Kawasaki 等人:“来自氨基戊梭菌的 H2O 形成 NADH 氧化酶的纯化和表征”Arch.Microbiol。
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    0
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Kawasaki S, Ishikura J, Chiba D, Nishino T, Youichi Niimura: "Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum : existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria."Archi Microbiol.
Kawasaki S、Ishikura J、Chiba D、Nishino T、Youichi Niimura:“氨基戊梭菌中形成 H2O 的 NADH 氧化酶的纯化和表征:专性厌氧细菌中存在氧解毒酶。”Archi Microbiol。
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    0
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Kouji Takeda, Yoshitaka Nishiyama, Koji Yoda, Toshihiro Watanabe, Kaori Nimura-Matune, Kiyoshi Mura, Chiyoko Tokue, Tetsuya Katob, Shinji Kawasaki, Youichi Niimura: "Distribution of Prx-linked Hydroperoxide Reductase Activity among Microorganismus"Biosci.
Kouji Takeda、Yoshitaka Nishiyama、Koji Yoda、Toshihiro Watanabe、Kaori Nimura-Matune、Kiyoshi Mura、Chiyoko Tokue、Tetsuya Katob、Shinji Kawasaki、Youichi Niimura:“微生物中 Prx 相关氢过氧化物还原酶活性的分布”Biosci。
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NIIMURA Youichi其他文献

NIIMURA Youichi的其他文献

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{{ truncateString('NIIMURA Youichi', 18)}}的其他基金

Development of Pro-biotic Lactic Acid Bacteria Scavenging Environmental Hydroperoxides
开发清除环境氢过氧化物的益生乳酸菌
  • 批准号:
    21580101
  • 财政年份:
    2009
  • 资助金额:
    $ 1.86万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Discovery of Food Microorganism : Fast and Effective Degradation of Intestinal Lipoperoxide and Hydrogen peroxide
食品微生物的发现:快速有效降解肠道过氧化脂和过氧化氢
  • 批准号:
    18580083
  • 财政年份:
    2006
  • 资助金额:
    $ 1.86万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Discovery of Food Microorganism : Fast and Effective Degradation of Intestinal Lipoperoxide
食品微生物的发现:快速有效地降解肠道过氧化脂质
  • 批准号:
    16580063
  • 财政年份:
    2004
  • 资助金额:
    $ 1.86万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
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