{"created":"2023-06-20T13:21:13.195678+00:00","id":1340,"links":{},"metadata":{"_buckets":{"deposit":"f23e4f78-6d33-485f-b41a-5b9fdc3a2cab"},"_deposit":{"created_by":1,"id":"1340","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1340"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001340","sets":["2:430:27"]},"author_link":["9642","9643","9644"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"山本, 宏"}],"nameIdentifiers":[{"nameIdentifier":"9642","nameIdentifierScheme":"WEKO"}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ヤマモト, ヒロシ"}],"nameIdentifiers":[{"nameIdentifier":"9643","nameIdentifierScheme":"WEKO"}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2000-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"     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.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第472号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"X2 分子生物機構論専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"1999"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"YAMAMOTO, Hiroshi","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"9644","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲472_要旨.pdf","filesize":[{"value":"375.2 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/1340/files/甲472_要旨.pdf"},"version_id":"52a92ff5-3a6d-456d-9800-e1b033b24eff"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria"},{"subitem_title":"Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["27"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1340","relation_version_is_last":true,"title":["Molecular-Biological and Physiological Characterization of Thioredoxin Peroxidase in Cyanobacteria"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:45:21.962116+00:00"}