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Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria
https://ir.soken.ac.jp/records/1340
https://ir.soken.ac.jp/records/13408e88b08a-5691-4776-85fe-08fd91c55912
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (375.2 kB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria | |||||
| タイトル | ||||||
| タイトル | Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者名 |
山本, 宏
× 山本, 宏 |
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| フリガナ |
ヤマモト, ヒロシ
× ヤマモト, ヒロシ |
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| 著者 |
YAMAMOTO, Hiroshi
× YAMAMOTO, Hiroshi |
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| 学位授与機関 | ||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||
| 学位名 | ||||||
| 学位名 | 博士（理学） | |||||
| 学位記番号 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | 総研大甲第472号 | |||||
| 研究科 | ||||||
| 値 | 生命科学研究科 | |||||
| 専攻 | ||||||
| 値 | X2 分子生物機構論専攻 | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 2000-03-24 | |||||
| 学位授与年度 | ||||||
| 値 | 1999 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | Active oxygen species (AOS), such as O<SUB>2</SUB><SUP>-</SUP>, ・OH and H<SUB>2</SUB>O<SUB>2</SUB>, are generated as a result of the incomplete reduction of O<SUB>2</SUB> during respiration and photosynthesis. Organisms living in oxygenic environments have an absolute requirement for mechanisms that detoxify AOS. AOS is extremely reactive and can cause severe damage of cell components in vitro, for example, by inactivating proteins, cleaving DNA and causing peroxidation of unsaturated fatty acids in cell membranes [1]. In plant chloroplasts, O<SUB>2</SUB><SUP>-</SUP>, the primary product in the oxygen reduction in chloroplasts, is immediately dismutated to H<SUB>2</SUB>O<SUB>2</SUB> and O<SUB>2</SUB> by superoxide dismutase. And H<SUB>2</SUB>0<SUB>2</SUB> is reduced to H<SUB>2</SUB>0 by ascorbate peroxidase, hich uses ascorbate as the electron donor. The univalently and divalently oxidized products of ascorbate, monodehydroascorbate and dehydroascorbate, respectively, are then re-reduced to ascorbate via the Halliwell-Asada pathway [2]．<br /> In cyanobacteria, H<SUB>2</SUB>O<SUB>2</SUB> is scavenged by peroxidases and/or catalases [3]. The peroxidases use electron generated during the photosynthetic electrons transport and such peroxidase activity is not observed in the presence of DCMU or in the dark． However, the physiological and enzymological properties of "light-dependent peroxidases" and the donors of electrons have not been fully clarified [4]．<br /> Several years ago, Kim et al. isolated a novel antioxidant enzyme, thioredoxin peroxidase (TPX), from yeast [5]. The enzyme catalyzes not only the reduction of H<SUB>2</SUB>O<SUB>2</SUB> to H<SUB>2</SUB>O but also the reduction of alkyl hydroperoxides to the corresponding alcohols and H<SUB>2</SUB>O, with thioredoxin as the electron donor [6,7]. Baier and Dietz reported that some plants have a gene for a homolog of TPX, bas1, and that the product of this gene is localized in the chloroplast stroma [8，9]. Furthermore, the genome of the cyanobacterium Synechocystis sp. PCC 6803 includes an open reading frame (ORF) designated sll0755 that encodes a putative homolog of TPX [9,10]. However, the existence of alkyl hydroperoxide reductase and TPX in cyanobacteria and chloroplasts has not been verified. Characterization of TPX in cyanobacteria will contribute to precise understanding of the detoxification of alkyl hydroperoxide in cyanobacteria and chloroplasts, and to our knowledge of the machinery of dissipation of excess photons as well as the water-water cycle in chloroplasts.<br /> The aims of the present study are: (1) biochemical identification of the product of ORF sll0755 from Synechocystis as TPX, and (2) characterization in vivo of the product of ORF sll0755 as a "light-dependent peroxidase" that reduces peroxides with electrons donated from the photosynthetic electron transport system.<br /><br /><br />Chapter 1. General introduction<br /> In this chapter, he reviewed the previous studies on peroxiredoxin in various organisms and the machineries and enzymes involved in the detoxification of active oxygen species in chloroplasts and cyanobacteria.<br /><br /><br />Chapter 2. Cloning of genes for thioredoxin peroxidase of cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 and characterization of the recombinant protein expressed in Escherichia coli<br /> In this chapter, he described the cloning of thioredoxin peroxidase genes (tpx) of cyanobacteria and biochemical characterization of recombinant TPX protein expressed in E. coli. The amino acid sequence deduced from the ORF designated sll0755 in Synechocystis is similar to the amino acid sequences of TPXs from other organisms. The product of ORF sll0755 was overexpressed as a fusion protein with a histidine tag in E. coli under the control of T7 promoter. The fusion protein purified by affinity chromatography showed the activity to reduce peroxides with thioredoxin and NADPH: thioredoxin oxidoreductase-coupling system from E. coli as an electron donor [11]. These results indicated that the ORF sll0755 encodes TPX. Furthermore, a 6.2-kb fragment of DNA that contained the tpx gene from Synechococcus was isolated by inverse PCR and normal PCR. The amino acid sequence deduced from the tpx gene from Synechococcus was also similar to those of TPXs from red algae and some land plants and conserved two catalytic cysteine residues.<br /><br /><br />Chapter 3. The function of thioredoxin peroxidase as a light-dependent peroxidase in Synechocystis sp. PCC 6803<br /> In this chapter, he described the targeted disruption of the tpx gene in Synechocystis cells and biochemical and physiological properties of the tpx<SUP>-</SUP> mutant. In cyanobacteria, H<SUB>2</SUB>O<SUB>2</SUB> is scavenged by peroxidase and/or catalase peroxidase [3]. Synechocystis has the activity of light-dependent peroxidase that reduces H<SUB>2</SUB>O<SUB>2</SUB> to water with electrons donated from the photosynthetic electron transport system. However, the functions of light-dependent peroxidase and its electron donor have not been clarified. The function of TPX as a "light-dependent peroxidase" in vivo has been examined by targeted disruption of the tpx gene in Synechocystis cells by insertional mutagenesis with a spectinomycin/streptomycin resistance gene cassette. tpx<SUP>-</SUP> cells were able to grow under low-intensity light (30 μmol photons m<SUP>-2</SUP> s<SUP>-1</SUP>), indicating that TPX is not essential for the growth of Synechocystis cells under non-oxidative conditions. In contrast to wild-type cells, the H<SUB>2</SUB>O<SUB>2</SUB>-dependent and tertiary-butyl hydroperoxide-dependent photosynthetic evolution of oxygen and the electron flow in photosystem II by adding H<SUB>2</SUB>O<SUB>2</SUB> or tertiary-butyl hydroperoxide to the cell suspension were absent in the cells of tpx<SUP>-</SUP> [11]. These results indicated that TPX functions as a "light-dependent peroxidase" whose activities are coupled to the photosynthetic electron transport system in Synechocystis cells. These findings provided for the first fine evidence that the TPX-dependent flow of electrons is operating in living photosynthetic cells. | |||||
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| 値 | 有 | |||||
