structural basis of the decamer architecture of a higher plant glutamine synthetase and substrate recognition mechanism

• Project/Area Number: 21570111
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: University of Yamanashi
• Principal Investigator: KUSUNOKI Masami 山梨大学, 大学院・医学工学総合研究部, 教授 (90135749)
• Co-Investigator(Renkei-kenkyūsha): HASE Toshiharu 大阪大学, たんぱく質研究所, 教授 (00127276)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2011: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2009: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 窒素同化 / 構造生物学 / 結晶構造 / グルタミン合成酵素 / 植物生理 / X線 / 酵素反応 / 活性部位 / 植物整理 / 触媒 / 結晶 / 立体構造 / 窒素代謝 / 植物生理学 / グルタミン合成酵

Research Abstract

We studied GS1a isoenzyme of maize glutamine synthetase in this research. Residues around Phe150 are responsible for interactions between two pentamers and we prepared many alanine-mutant enzymes for these residues. As a result we found the alanine mutants with high Km values for residues at the location spanning the two-pentamer interaction region and the substrate glutamic acid entry region into the catalytic site. The former region is distant from the catalytic site and we obtained two good crystals of G241A and W243A mutant enzymes at 2. 10 A and 2. 80 A resolutions, respectively there. These crystal structures revealed that there is a region on the enzyme involved in the substrate affinity distinct from the catalytic site.

Research Products

• [Journal Article] Galactose recognition by a tetrameric C-type lectin, CEL-LV, containina the EPN carbohydrate-recognition motif (2011)
  • Author(s): Tomomitsu Hatakeyama
  • Journal Title: J. Biol. Chem. Volume: 583 Pages: 219-223
  • Peer Reviewed
• [Journal Article] Production of tetraketide lactones by mutated Antirrhinum majus chalcone synthases (AmCHS1) (2010)
  • Author(s): Masayoshi Hatayama
  • Journal Title: J.Biosci.Bioeng. Volume: 110 Pages: 158-164
  • Peer Reviewed
• [Journal Article] Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitiveinhibitor maltose (2010)
  • Author(s): Keizo Yamamoto
  • Journal Title: FEBS Journal Volume: 227 Pages: 4205-4214
  • Peer Reviewed
• [Journal Article] Structural basis for chiral substrate recognition by two 2, 3-butanediol dehydrogenases. (2010)
  • Author(s): Masato Otagiri
  • Journal Title: FEBS Letters 584 Pages: 219-223
  • Peer Reviewed
• [Presentation] Distant residues of maize glutamine synthetase affecting substrate affinity (2011)
  • Author(s): M. Kusunoki, T. Ozaki, A. Nakagawa, T. Hase
  • Organizer: 国際結晶学会
  • Place of Presentation: マドリード,スペイン
  • Year and Date: 2011-08-27
• [Presentation] トウモロコシ由来グルタミン合成酵素GS1aとGS1d活性の変化の構造学的研究 (2011)
  • Author(s): 尾崎健・中川敦史・長谷俊治・小林大千・楠木正巳
  • Organizer: 日本結晶学会年会
  • Place of Presentation: 北海道大学
• [Presentation] トウモロコシ由来グルタミン合成酵素GS1aの1-Ring変異体F150Vの結晶構造 (2010)
  • Author(s): 尾崎健
  • Organizer: 日本結晶学会
  • Place of Presentation: 関西学院大学 兵庫県
  • Year and Date: 2010-12-06
• [Presentation] Mutational study of maize glutamine synthetase(GS1a) : Identification of two distinct sites necessary for high affinity for glutamate (2010)
  • Author(s): 尾崎健
  • Organizer: 国際植物窒素同化代謝会議
  • Place of Presentation: 犬山国際観光センターフロイデ
  • Year and Date: 2010-07-28
• [Presentation] トウモロコシ由来グルタミン合成酵素GS1aの変異体G241AとW243Aの結晶構造解析 (2010)
  • Author(s): 尾崎健・中川敦司・長谷俊治・楠木正巳
  • Organizer: 日本結晶学会
  • Place of Presentation: 大阪大学コンベンションセンター
• [Presentation] Mutational study of maize glutamine synthetase(GS1a): Identification of two distinct sites necessary for high affinity for glutamate (2010)
  • Author(s): T. Ozaki, M. Yamada, G. Estivill, A. J. Marquez, A. Nakagawa, M. Kusunoki and T. Hase
  • Organizer: International Symposium on the Nitrogen Nutrition of Plants
  • Place of Presentation: 犬山
• [Presentation] 植物グルタミン合成酵素の基質認識機構の研究:グルタミン酸への親和性を決定する活性中心遠位領域の同定 (2009)
  • Author(s): 尾崎健・楠木正巳・高木淳一・中川敦司・長谷俊治
  • Organizer: 日本生化学会大会
  • Place of Presentation: 神戸ポートアイランド
• [Remarks]
  • URL: http://www.ab11.yamanashi.ac.jp/ABF/No2/Kusu_Lab/research.html
• [Remarks]
  • URL: http://www.abll.yamanashi.ac.jp/ABF/No2/Kusu_Lab/GS.html
• [Remarks]
  • URL: http://www.ab11.yamanashi.ac.jp/ABF/No2/Kusu_Lab/GS.html
• [Remarks]
  • URL: http://www.abll.yamanashi.ac.jp/ABF/No2/Kusu_Lab/GS.html