Elucidation of catalytic mechanism of two characteristic enzymes and its application based on crystal structures.

• Project/Area Number: 24570130
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Kyoto University
• Principal Investigator: FUJIHASHI Masahiro 京都大学, 理学(系)研究科(研究院), 助教 (10397581)
• Co-Investigator(Renkei-kenkyūsha): SATO Tsutomu 新潟大学, 農学部, 准教授 (80334655) ; ISHIDA Toyokazu 独立行政法人産業技術総合研究所, ナノシステム研究部門, 研究員 (70443166)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 基質の歪み / 脱炭酸反応 / 結晶構造解析 / 高分解能結晶構造 / 計算機シミュレーション / 酵素反応機構 / ODCase / OMPDC / OMPDCase / X線結晶構造 / 国際情報交換 / X線結晶構造解析 / 酵素反応 / 結晶構造

Outline of Final Research Achievements

This project targeted two enzymes. Regarding the first enzyme ODCase, we determined about 10 crystal structures using various mutants and/or ligands. The best resolution of the determined structures is 1.03 A, which is much better than those of the over 200 determined ODCase structures thus far. Based on the structures with biochemical and computational analyses, we elucidated that substrate distortion contributes to 10-15% free energy decrease of the transition state by ODCase. Regarding the other enzyme Cyc2, we elucidated the enzyme is not appropriate for the crystallographic analysis. We are now attempt to purify and crystallize a homologous protein from a different organism.

Research Products

• [Journal Article] 結晶構造を基にした酵素反応機構の解析 ～オロチジン一リン酸脱炭酸酵素を題材に～ (2015)
  • Author(s): 藤橋雅宏
  • Journal Title: 日本結晶学会誌 Volume: 56 Pages: 236-240
  • NAID: 130004688355
  • Peer Reviewed
• [Journal Article] Orotidine monophosphate decarboxylase - A fascinating workhorse enzyme with therapeutic potential (2015)
  • Author(s): Masahiro Fujihashi, Jagjeet S. Mnpotra, Emil F. Pai and Lakshmi P. Kotra
  • Journal Title: Journal of Genetics and Genomics Volume: 未定
  • Peer Reviewed
• [Journal Article] Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase (2013)
  • Author(s): 藤橋雅宏、石田 豊和、黒田新悟、Lakshmi P. Kotra, Emil F. Pai, 三木邦夫
  • Journal Title: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Volume: 135巻 Issue: 46 Pages: 17432-17443
  • DOI: 10.1021/ja408197k
  • Peer Reviewed
• [Journal Article] Atomic Resolution Structure of the Orotidine 5'-Monophosphate Decarboxylase Product Complex Combined with Surface Plasmon Resonance Analysis: IMPLICATIONS FOR THE CATALYTIC MECHANISM. (2013)
  • Author(s): Masahiro Fujihashi, Kazuya Mito, Emil F. Pai, Kunio Miki
  • Journal Title: J. Biol. Chem. Volume: 288 Issue: 13 Pages: 9011-9016
  • DOI: 10.1074/jbc.m112.427252
  • Peer Reviewed
• [Presentation] オロチジン一リン酸脱炭酸酵素の反応機構における基質歪みの役割 (2014)
  • Author(s): 藤橋雅宏, 石田豊和, 黒田新悟, 三登一八, Emil F. Pai, Lakshmi P. Kotra, 三木邦夫
  • Organizer: 日本結晶学会
  • Place of Presentation: 東京大学農学部
  • Year and Date: 2014-11-01
• [Presentation] Substrate distortion in the catalysis of orotidine monophosphate decarboxylase (2014)
  • Author(s): Masahiro Fujihashi, Toyokazu Ishida, Shingo Kuroda, Kazuya Mito, Lakshmi P. Kotra, Emil F. Pai, Kunio Miki
  • Organizer: 23rd Congress and General Assembly of the International Union of Crystallography
  • Place of Presentation: Montreal, Quebec, Canada
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] オロチジン一リン酸脱炭酸酵素の基質の歪みを利用した反応機構 (2014)
  • Author(s): 藤橋 雅宏, 石田 豊和, 黒田 新悟, 三登 一八, Lakshmi P. Kotra, Emil F. Pai, 三木 邦夫
  • Organizer: 第14回 日本蛋白質科学会年会
  • Place of Presentation: 横浜市 ワークピア横浜/横浜産貿ホール マリネリア
  • Year and Date: 2014-06-27
• [Presentation] オロチジン一リン酸脱炭酸酵素の反応機構 (2014)
  • Author(s): 藤橋雅宏, 石田豊和, 黒田新悟, 三登一八, Emil F. Pai, Lakshmi P. Kotra, 三木邦夫
  • Organizer: 物構研サイエンスフェスタ2013
  • Place of Presentation: つくば市 つくば国際会議場
• [Presentation] オロチジン一リン酸脱炭酸酵素の反応触媒機構の解析 (2013)
  • Author(s): 藤橋雅宏, 石田豊和, 黒田新悟, 三登一八, Emil F Pai, Lakshmi P. Kotra, 三木邦夫
  • Organizer: 第13回日本蛋白質科学会年会
  • Place of Presentation: 鳥取市 とりぎん文化会館
• [Presentation] 基質構造の歪みを利用するオロチジン一リン酸脱炭酸酵素の反応機構 (2012)
  • Author(s): 藤橋雅宏, 石田豊和, 黒田新悟, Emil F Pai, Lakshmi P. Kotra, 三木邦夫
  • Organizer: 平成24年度日本結晶学会年会
  • Place of Presentation: 東北大学片平キャンパス (仙台市)