Structural characterization of reaction pathway in a PLP-enzyme, methionine gamma-lyase: crystallographic snapshots of key intermediates

• Project/Area Number: 23370050
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Kyoto Institute of Technology
• Principal Investigator: HARADA shigeharu 京都工芸繊維大学, 工芸科学研究科, 教授 (80156504)
• Project Period (FY): 2011-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥14,170,000 (Direct Cost: ¥10,900,000、Indirect Cost: ¥3,270,000); Fiscal Year 2014: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2013: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2012: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2011: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
• Keywords: 酵素反応機構 / 生体分子 / PLP酵素 / タンパク質 / 時分割X線解析 / スナップショット構造 / 酸素 / 蛋白質

Outline of Final Research Achievements

Pyridoxal 5'-phosphate (PLP) is an indispensable cofactor in many enzymes. The catalytic mechanism of the first part, from substrate binding to PLP to formation of quinonid intermediate, is characterized in several PLP enzymes, but the latter half is not fully understood. We determined X-ray structures of a series of reaction intermediates to elucidate the mechanism of PLP-dependent methionine γ-lyase. The structures reveal that the methionine degradation is carried out by aldimine-based C4'-Nα-Cα system, followed by proton migration from the Cα to C4' position. After releasing methanethiol, the latter half of intermediates suggest that the reaction processes leading to the releasing 2-aminobutyrate accounted for a concerted [1,5]-hydrogen sigmatropy with respect to the C4' and Cγ-carbon sites. Taken together, these structural intermediates successfully depicted overall reaction process of γ-elimination.

Research Products

• [Journal Article] Anemia amelioration by lactose infusion during trypanosomiasis could be associated with erythrocytes membrane de-galactosylation (2014)
  • Author(s): Balogun, EO., Balogun, JAB., Yusuf, S., Inuwa, HM., Ndams, IS., Sheridan, P., Inaoka, DK., Shiba, T., Harada, S., Kita, K., Esievo, KAN., Nok AJ.
  • Journal Title: Veterinary Parasitology Volume: 199 Issue: 3-4 Pages: 259-263
  • DOI: 10.1016/j.vetpar.2013.10.013
  • Peer Reviewed
• [Journal Article] Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase (2014)
  • Author(s): Emmanuel Oluwadare Balogun, Daniel Ken Inaoka, Tomoo Shiba, Yasutoshi Kido, Chiaki Tsuge, Takeshi Nara, Takashi Aoki, Teruki Honma, Akiko Tanaka, Masayuki Inoue, Shigeru Matsuoka, Paul A.M. Michels, Kiyoshi Kita, Shigeharu Harada
  • Journal Title: Molecular Microbiology Volume: 94 Issue: 6 Pages: 1315-1329
  • DOI: 10.1111/mmi.12831
  • Peer Reviewed
• [Journal Article] Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis (2014)
  • Author(s): Luke Young, Benjamin May, Alice Pendlebury-Watt, Julia Shearman, Catherine Elliott, Mary S. Albury, Tomoo Shiba, Daniel Ken Inaoka, Shigeharu Harada, Kiyoshi Kita, Anthony L. Moore
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Bioenergetics Volume: 1837 Issue: 7 Pages: 1219-1225
  • DOI: 10.1016/j.bbabio.2014.01.027
  • Peer Reviewed
• [Journal Article] Identification and Characterization of Sialidase-Like Activity in the Developmental Stages of Amblyomma Variegatum (2013)
  • Author(s): Audu J. Natala, Emmanuel 0. Balogun, Joshua A. B. Balogun, Hajiya M. Inuwa, Andrew J. Nok, Tomoo Shiba, Shigeharu Harada,Kiyoshi Kita, Rowland l. S. Agbede, King A. N. Esievo
  • Journal Title: Journal of Medical Entomology Volume: 50 (1) Issue: 1 Pages: 85-93
  • DOI: 10.1603/me12152
  • Peer Reviewed
• [Journal Article] Structure of the trypanosome cyanide-insensitive alternative oxidase (2013)
  • Author(s): Tomoo Shiba, Yasutoshi Kido, Kimitoshi Sakamoto, Daniel Ken Inaoka, Chiaki Tsuge, Ryoko Tatsumi,Gen Takahashi, Emmanuel Oluwadare Balogun, Takeshi Nara, Takashi Aoki, Teruki Honma, Akiko Tanaka, Masayuki Inoue, Shigeru Matsuoka, Hiroyuki Saimoto, Anthony L. Moore, Shigeharu Harada, Kiyoshi Kita
  • Journal Title: Proc. Nat1. Acad. Sci. USA Volume: 110 (12) Issue: 12 Pages: 4580-4585
  • DOI: 10.1073/pnas.1218386110
  • Peer Reviewed
• [Journal Article] Diversity of parasite complex II (2013)
  • Author(s): Shigeharu Harada, Daniel Ken Inaoka, Junko Ohmori, Kiyoshi Kita
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Bioenergetics Volume: 1827 Issue: 5 Pages: 658-667
  • DOI: 10.1016/j.bbabio.2013.01.005
  • Peer Reviewed
• [Journal Article] Unraveling the Heater: New Insights into the Structure of the Alternative Oxidase (2013)
  • Author(s): Anthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita, Kikukatsu Ito
  • Journal Title: Annual Review of Plant Biology Volume: 64 Issue: 1 Pages: 637-663
  • DOI: 10.1146/annurev-arplant-042811-105432
  • Peer Reviewed
• [Journal Article] The alternative oxidases: simple oxidoreductase proteins with complex functions (2013)
  • Author(s): Luke Young, Tomoo Shiba, Shigeharu Harada, Kiyoshi Kita, Mary S. Albury, Anthony L. Moore
  • Journal Title: Biochem. Soc. Trans. Volume: 41 Issue: 5 Pages: 1305-1311
  • DOI: 10.1042/bst20130073
  • Peer Reviewed
• [Journal Article] The Role of Amino Acid Residues in the Active Site of <small>L</small>-Methionine &#947;-lyase from <i>Pseudomonas putida</i> (2012)
  • Author(s): Fukumoto, M.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 76 Issue: 7 Pages: 1275-1284
  • DOI: 10.1271/bbb.110906
  • NAID: 130001862713
  • ISSN: 0916-8451, 1347-6947
  • Peer Reviewed
• [Journal Article] A Conserved Lysine Residue in the Crenarchaea-Specific Loop is Important for the Crenarchaeal Splicing Endonuclease Activity (2011)
  • Author(s): Maho Okuda, Tomoo Shiba, Daniel-Ken Inaoka, Kiyoshi Kita, Genji Kurisu, Shigeru Mineki, Shigeharu Harada, Yoh-ichi Watanabe, Shigeo Yoshinari
  • Journal Title: J.Mol.Biol. Volume: 405 Pages: 92-104
  • Peer Reviewed
• [Journal Article] A Drosophila model for the screening of bioavailable NADPH oxidase inhibitors and antioxidants (2011)
  • Author(s): Nguyen Thi Tu Anh, Maiko Nishitani, Shigeharu Harada, Masamitsu Yamaguchi, Kaeko
  • Journal Title: Mol Cell Biochem. Volume: 352 Pages: 91-98
  • Peer Reviewed
• [Journal Article] Essential Role of Duox in Stabilization of Drosophila Wing (2011)
  • Author(s): Nguyen Thi Tu Anh, Maiko Nishitani, Shigeharu Harada, Masamitsu Yamaguchi, Kaeko Kamei
  • Journal Title: J.Biol.Chem. Volume: 286 Pages: 33244-33251
  • Peer Reviewed
• [Presentation] 高度好熱菌Sulfolobus tokodai由来シスタチオニン合成酵素の結晶構造 (2014)
  • Author(s): 川村あやか、花田尚子、山田哲也、田村隆、佐藤暖、志波智生、稲垣賢二、原田繁春
  • Organizer: 第８７回日本生化学会大会
  • Place of Presentation: 京都市
  • Year and Date: 2014-10-16
• [Presentation] Pseudomonas putida由来メチオニンγ-リアーゼの結晶構造解析 (2014)
  • Author(s): 湯之戸俊介、安田江里、古谷和大、福本充樹、工藤大蔵、志波智生、佐藤暖、田村隆、原田繁春、稲垣賢二
  • Organizer: 第８７回日本生化学会大会
  • Place of Presentation: 京都市
  • Year and Date: 2014-10-16
• [Presentation] 赤痢アメーバ由来メチオニンγ-リアーゼの構造生物学的研究 (2014)
  • Author(s): 坂井菜摘、近藤輝、岡知宏、唐木剛、村野祥子、野崎智義、佐藤暖、原田繁春、志波智生
  • Organizer: 第８７回日本生化学会大会
  • Place of Presentation: 京都市
  • Year and Date: 2014-10-16
• [Presentation] Crystal structures of Pseudomonas putida L-methionine γ-lyase and its C116H mutant enzyme complexed with substrate: Insights into the substrate recognition mechanism (2014)
  • Author(s): Eri Yasuda, Mitsuki Fukumoto, Kazuo Furutani, Syunsuke Yunoto, Takashi Tamura, Tomoo Shiba, Dan Sato, Shigeharu Harada and Kenji Inagaki
  • Organizer: The Fourth International Conference on Cofactors
  • Place of Presentation: イタリア パルマ市
  • Year and Date: 2014-08-25 – 2014-08-28
• [Presentation] 赤痢アメーバ由来メチオニンγリアーゼ２の結晶構造解析 (2014)
  • Author(s): 近藤 輝、酒井菜摘、岡 知宏、村、唐木 剛、野崎 智義、佐藤 暖、志波 智生、原田 繁春
  • Organizer: 第１４回日本蛋白質科学会年会
  • Place of Presentation: 横浜市
  • Year and Date: 2014-06-26
• [Presentation] Pseudomonas putida由来メチオニンγリアーゼの結晶構造解析 (2014)
  • Author(s): 湯之戸俊介、古谷和大、福本充樹、工藤大蔵、志波智生、佐藤暖、田村隆、 原田繁春、稲垣賢二
  • Organizer: 第１４回日本蛋白質科学会年会
  • Place of Presentation: 横浜市
  • Year and Date: 2014-06-25
• [Presentation] L-メチオニン γ-リアーゼ基質複合体のX線結晶構造解析と基質認識に関わる残基の機能解析 (2014)
  • Author(s): 安田江里，古谷和大，佐藤暖，福本充樹，工藤大蔵，志波智生，原田繁春，田村隆，稲垣賢二
  • Organizer: 日本ビタミン学会第６６回大会
  • Place of Presentation: 姫路市
  • Year and Date: 2014-06-13 – 2014-06-14
• [Presentation] 赤痢アメーバ由来メチオニンγ-リアーゼの結晶学的構造解析 (2013)
  • Author(s): 酒井菜摘、岡知宏、唐木剛、村野祥子、近藤輝、佐藤暖、野崎智義、原田繁春、志波智生
  • Organizer: 第８６回日本生化学会大会
  • Place of Presentation: 横浜
• [Presentation] 好熱好酸性アーキアSulfolobus tokodaii由来シスタチオニン γ‐シンターゼの構造解析 (2013)
  • Author(s): 清水あやか花田尚子、山田哲也、田村隆、佐藤暖、志波智生、稲垣賢二、原田繁春
  • Organizer: 第８６回日本生化学会大会
  • Place of Presentation: 横浜
• [Presentation] Pseudomonas putida由来メチオニン γ-リアーゼの結晶構造解析 (2013)
  • Author(s): 古谷和大、湯之戸俊介、福本充樹、工藤大蔵、志波智生、佐藤暖、田村隆、原田繁春、稲垣賢二
  • Organizer: 第８６回日本生化学会大会
  • Place of Presentation: 横浜
• [Presentation] 赤痢アメーバ由来メチオニンγ -リアーゼの精製と結晶構造解析 (2013)
  • Author(s): 酒井菜摘、岡知宏、村野祥子、唐木剛、野崎智義、佐藤暖、志波智生、原田繁春
  • Organizer: 第１３回日本蛋白質科学会年会
  • Place of Presentation: 鳥取
• [Presentation] 好熱好酸性アーキアSulfolobus tokodaii由来シスタチオニン合成酵素のX線結晶構造解析 (2013)
  • Author(s): 清水あやか、花田尚子、山田哲也、田村隆、志波智生、稲垣賢二、原田繁春
  • Organizer: 第１３回日本蛋白質科学会年会
  • Place of Presentation: 鳥取
• [Presentation] 好熱好酸性アーキアSulfolobus tokodaii由来シスタチオニンγ-シンターゼのX線結晶構造解析 (2012)
  • Author(s): 花田尚子
  • Organizer: 第85回日本生化学会大会
  • Place of Presentation: 福岡
• [Presentation] 好熱好酸性アーキアSulfolobus tokodaii 由来シスタチオニンγ-シンターゼの活性中心残基残基の機能解析 (2012)
  • Author(s): 山田哲也
  • Organizer: 第85回日本生化学会大会
  • Place of Presentation: 福岡
• [Presentation] トリパノソーマクルージのジヒドロオロト酸脱水素酵素に対する選択的阻害剤の設計 (2011)
  • Author(s): 稲岡ダニエル健、飯田舞子、田淵俊行、松岡茂、志波智生、坂元君年、鈴木重雄、奈良武司、青木孝、井上将行、本間光貴、田仲昭子、原田繁春、北潔
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-24
• [Presentation] Structural analysis of glycerol kinase from human African trypanosomes for anti-trypanosomal drug design (2011)
  • Author(s): Balogun Emmanuel O.、稲岡ダニエル健、志波智生、城戸康年、奈良武司、青木孝、本間光貴、田仲昭子、井上将行、松岡茂、Michels Paul AM.、原田繁春、北潔
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-24
• [Presentation] 寄生虫ミトコンドリア呼吸鎖膜タンパク質の立体構造と論理的阻害剤設計 (2011)
  • Author(s): 原田繁春、稲岡ダニエル、松岡茂、志波智生、坂元君年、奈良武司、青木孝、井上将行、本間光貴、田仲昭子、北潔
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-23
• [Presentation] Pseudomonas putida由来L-メチオニンγ-リアーゼの活性中心残基の機能解析 (2011)
  • Author(s): 山田哲也、福本充樹、工藤大蔵、原田繁春、田村隆、稲垣賢二
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-23
• [Presentation] クルーズトリパノソーマ由来アスパラギン酸トランスカルバモイラーゼと阻害剤複合体のX線結晶解析 (2011)
  • Author(s): 藤田康介、的場一晃、奈良武司、青木孝、幸瞳、本間光貴、田仲昭子、松高祥子、松岡茂、井上将行、志波智生、稲岡ダニエル健、北潔、原田繁春
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-22
• [Presentation] トキソプラズマ原虫の活性型ヌクレオシド三リン酸加水分解酵素の構造 (2011)
  • Author(s): 濱崎遥香、的場一晃、志波智生、竹内勤、浅井隆志、原田繁春
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-22
• [Presentation] 赤痢アメーバメチオニンγ-リアーゼ1の結晶中で捉えたα,γ-脱離反応機構 (2011)
  • Author(s): 岡知宏、佐藤暖、村野祥子、唐木剛、中沢隆、野崎智義、原田繁春
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-22
• [Presentation] トリパノソーマ原虫のミトコンドリアの内膜に局在する「シアン耐性酸化酵素Trypanosome Alternative Oxidase (TAO)」の結晶構造 (2011)
  • Author(s): 志波智生、城戸康年、稲岡ダニエル健、坂元君年、奈良武司、青木孝、本間光貴、田仲昭子、井上将行、松岡茂、斎本博之、ムーア・アンソニー、原田繁春、北潔
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都(国立京都国際会館)
  • Year and Date: 2011-09-21
• [Presentation] Structure based design and development of inhibitors with high specificity and inhibitory activity against mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum (2011)
  • Author(s): S.Harada
  • Organizer: IUMS : International Congress of Bacteriology and Applied Microbiology (BAM), and the International Congress of Mycology
  • Place of Presentation: 札幌(札幌コンベンションセンター)(招待講演)
  • Year and Date: 2011-09-08
• [Presentation] クルーズトリパノソーマ由来アスパラギン酸トランスカルバモイラーゼと阻害剤との複合体構造 (2011)
  • Author(s): 的場一晃、藤田康介、奈良武司、青木孝、幸瞳、本間光貴、田仲昭子、松高祥子、松岡茂、井上将行、志波智生、稲岡ダニエル健、北潔、原田繁春
  • Organizer: 第11回日本蛋白質科学会年会
  • Place of Presentation: 大阪(ホテル阪急エキスポパーク)
  • Year and Date: 2011-06-09
• [Presentation] トキソプラズマ原虫由来NTPaseの活性型変異体の構造 (2011)
  • Author(s): 濱崎遥香、的場一晃、志波智生、竹内勤、浅井隆志、原田繁春
  • Organizer: 第11回日本蛋白質科学会年会
  • Place of Presentation: 大阪(ホテル阪急エキスポパーク)
  • Year and Date: 2011-06-08
• [Presentation] 赤痢アメーバメチオニンγ-リアーゼ1の結晶中で捉えたα,γ-脱離反応進行に伴うスナップショット構造 (2011)
  • Author(s): 岡知宏、佐藤暖、村野祥子、唐木剛、中沢隆、野崎智義、原田繁春
  • Organizer: 第11回日本蛋白質科学会年会
  • Place of Presentation: 大阪(ホテル阪急エキスポパーク)
  • Year and Date: 2011-06-08