Alternative TitleSynthetic Approach toward Elucidation of the Structure and Function of Phytochromes
Periodical title平成18(2006)年度 科学研究費補助金 基盤研究(B) 研究成果報告書 = 2006 Fiscal Year Final Research Report
Note (General)植物内に存在する光受容色素蛋白質フィトクロムは,生理学的に不活性で赤色光吸収型のPrと生理学的に活性で遠赤色光吸収型のPfrとの間で可逆的に相互変換する光分子スイッチ機能を有する。この「赤・遠赤色光可逆的反応」は,植物の発生や成長・分化などの様々な過程に広く関係し,環境の光情報を植物に伝える重要な役割を果たしており,植物が生きて行くために欠くことのできない調節作用の一つである。本研究では,発色団の構造と再構成フィトクロムの機能との間の相関など,この分野を飛躍的に発展させるために,これまで合成が困難とされてきたビリン系発色団の効率的合成法及び立体化学を固定した誘導体の新合成法の確立など,フィトクロムの構造と機能の解明に関する有機合成化学的アプローチを推進し,次のような研究成果を挙げることができた。1.アグロバクテリオフィトクロムAgp1のアポ蛋白質に対する発色団ビリベルジン(BV)の共有結合部位を解明するために,種々のBV誘導体を合成し,再構成実験によりA環3位のビニル基で結合していることを明らかにした。2.AB環及びCD環部位の立体配置及び配座をそれぞれ固定した可能な4種のBV誘導体を全て合成し,Agp1及びAgp2のアポ蛋白質と再構成させて得られる人工フィトクロムと天然型フィトクロムのスペクトル的ならびに生化学的比較研究により,Pr型発色団の15位の立体化学はZ-anti, Pfr型はE-antiであり,Pr型の5位の立体化学はZ-synであることを世界で初めて直接的に解明した。3.15位をZ-antiで固定した発色団とアポ蛋白質を再構成させて得られるホロ蛋白質の良質な単結晶を得ることに成功し,X線構造解析を目指して大きく前進した。4.分子量12万の植物フィトクロム蛋白質を高純度で単離精製するために,修飾型発色団を担体に結合させたアフィニティークロマトグラフィーの実現に向けて大きく前進した。5.バクテリオフィトクロムAgp1の光異性化の詳細な反応機構とシグナル伝達機構の解明を目指して,AB環及びCD環部分の立体化学を二重に固定したBV発色団の合成を可能にした。6.光学活性な発色団を合成するために,これまで本プロジェクトとは独立に開発してきた酒石酸エステルを不斉源として活用する高効率・高選択的なエナンチオ選択的反応を開発するとともに,「シン効果」に関する重要な知見を得た。
Phytochromes are photoreceptors existing in plants and carry a bilin chromophore, which is covalently attached to the protein. Phytochromes behave as photoreversible molecular switches between physiologically inactive red light-absorbing Pr and physiologically active far-red light-absorbing Pfr. This "red-far-red lights photoreversible reaction" plays important role to sense light information in the environment and related to various processes such as development, growth, and differentiation of plants. In the present research project on phytochromes, we established the efficient methods for the preparation of bilin chromophores and their sterically locked derivatives, which were assembled with apoproteins of phytochromes. The following are the results obtained by synthetic organic approach toward elucidation of the structure and function of phytochromes1. Many bacterial phytochromes carry biliverdin (BV) as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agpl of Agrobacterium tumefaciens, BV is covalently attached to a cysteine residue close to the N-terminus (position 20). By testing different natural and synthetic BV derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment.2. Total syntheses of BV derivatives with sterically locked AB-or CD-ring component were accomplished via new and efficient methods towards elucidation of the stereochemistry of the chromophore in phytochromes. The chromophores were tested for assembly with Agrobacterium phytochrome Agpl and Agp2, which incorporate BV as natural chromophore. It was found that the chromophore of Agpl adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form, and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agpl. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.3. The N-terminal photosensory module of Agrobacterium phytochrome Agpl, Agp1-M15, was assembled with a sterically locked synthetic BV-derivative 15Za to give crystals of 15Za-Agpl-M15, which diffract to 3.4 A resolution and belong to the tetragonal space group 1422 with unit cell dimensions a=b=174 A, c=80 A.4. A new type of affinity chromatography was investigated to establish an efficient method for the isolation of plant phytochromes by attaching synthetic chromophores as a ligand on carrier resin.5. Chromophores doubly locked at AB-and CD-rings were synthesized to determine the stereochemistry of AB-ring component of Pfr and also toward analysis of the detailed mechanism of photoisomerization of Agpl and signal transduction.6. To exploit an efficient method for the preparation of optically active phytochrome chromophores, new highly efficient and enantioselective reactions were developed utilizing tartaric acid esters as a chiral auxiliary. Furthermore new insights were obtained regarding "syn-effect".
研究課題/領域番号:15350021, 研究期間(年度):2003-2006
出典：「フィトクロムの構造と機能解明のための有機合成化学的アプローチ」研究成果報告書 課題番号15350021 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
Sourcehttps://kanazawa-u.repo.nii.ac.jp/?action=repository_action_common_download&item_id=43022&item_no=1&attribute_id=26&file_no=1
Related Materialhttps://kaken.nii.ac.jp/search/?qm=50110599
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15350021/
https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-15350021/153500212006kenkyu_seika_hokoku_gaiyo/
Data Provider (Database)国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）