Elucidation of catalytic mechanisms as well as subunit-assembly of nitrile hydratase family enzymes by using advanced structural studies

• Project/Area Number: 24350082
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Chemistry related to living body
• Research Institution: Akita University (2014); Tokyo University of Agriculture and Technology (2012-2013)
• Principal Investigator: ODAKA Masafumi 秋田大学, 工学(系)研究科(研究院), 教授 (20224248)
• Co-Investigator(Renkei-kenkyūsha): KUROKI Ryota 独立行政法人日本原子力研究開発機構, 量子ビーム応用研究部門, ユニット長 (30391246) ; YOHDA Masafumi 東京農工大学, 大学院工学研究院, 教授 (50250105)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥18,460,000 (Direct Cost: ¥14,200,000、Indirect Cost: ¥4,260,000); Fiscal Year 2014: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2013: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2012: ¥7,280,000 (Direct Cost: ¥5,600,000、Indirect Cost: ¥1,680,000)
• Keywords: 時間分割結晶構造解析 / 翻訳後修飾 / システインスルフェン酸 / 酵素反応 / 触媒機構 / 反応中間体 / 水和反応 / 結晶構造 / 酵素触媒機構 / 金属酵素 / 構造生物学 / 中性子構造解析 / 酵素反応機構 / 時分割結晶構造解析

Outline of Final Research Achievements

Catalytic reaction of a Fe-type nitrile hydratase betaR56K mutant was studied by time-resolved crystallography. The results showed that the metal-coordinated substrate is nucleophilically attacked by the O(SO-) atom of alphaCys114-SO-;, followed by nucleophilic attack of the S(SO-) atom by a betaArg56-activated water molecule to release the product amide and regenerate alphaCys114-SO-;. Also, kinetic as well as crystallographic studies on the mutant SCNases revealed that both NHase and SCNase share the catalytic mechanism and that the size and shape of their substrate binding pockets are predominantly control the substrate selectivity.

Research Products

• [Journal Article] Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the β-carbonic anhydrase family enzymes (2013)
  • Author(s): Ogawa T, Noguchi K, Saito M, Nagahata Y, Kato H, Ohtaki A, Nakayama H, Dohmae N, Matsushita Y, Odaka M, Yohda M, Nyunoya H, Katayama Y
  • Journal Title: J. Am. Chem. Soc. Volume: 135 Issue: 10 Pages: 3818-3825
  • DOI: 10.1021/ja307735e
  • Peer Reviewed
• [Journal Article] Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase (2013)
  • Author(s): Yasuaki Yamanaka, Takatoshi Arakawa, Toshinori Watanabe, Satoshi Namima, Masa Sato, Shota Hori, Akashi Ohtaki, Keiichi Noguchi, Yoko Katayama, Masafumi Yohda, Masafumi Odaka
  • Journal Title: Journal of Bioscience and Bioengineering Volume: Vol. 116、Issue 1 Issue: 1 Pages: 22-27
  • DOI: 10.1016/j.jbiosc.2013.01.013
  • NAID: 110009634740
  • Peer Reviewed
• [Journal Article] Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase. (2013)
  • Author(s): Yamanaka Y, Arakawa T, Watanabe T, Namima S, Sato M, Hori S, Ohtaki A, Noguchi K, Katayama y Yohda M, Odaka M.
  • Journal Title: J Biosci Bioeng.
  • NAID: 110009634740
  • Peer Reviewed
• [Presentation] Reaction mechanism of nitrile hydratase proposed by the high resolution transition state structure of the enzyme-substrate complex (2014)
  • Author(s): Yasuaki Yamanaka, Yuki Kato, Koichi Hashimoto, Keisuke Iida, Kazuo Nagasawa, Hiroshi Nakayama, Naoshi Dohmae, Keiichi Noguchi, Takumi Noguchi, Masafumi Yohda, Masafumi Odaka
  • Organizer: 7th Asian Biological Inorganic Chemistry Conference
  • Place of Presentation: Surfers Paradise, Gold Coast
  • Year and Date: 2014-11-30 – 2014-12-05
• [Presentation] 活性化タンパク質P15Kによるチオシアネート加水分解酵素成熟化機構の解明 (2012)
  • Author(s): 渡邉俊則、中西拓也、荒川孝俊、堀祥太、中山洋、片山葉子、堂前直、Gumataotao Natarie、野口恵一、養王田正文、尾高雅文
  • Organizer: 第85回日本生化学会大会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 2012-12-16
• [Presentation] Rhodococcus erythropolis N771由来ニトリルヒドラターゼの触媒機構における必須アルギニン残基の機能 (2012)
  • Author(s): 相原敬之、山中保明、野口恵一、中山洋、堂前直、養王田正文、尾高雅文
  • Organizer: 第85回日本生化学会大会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 2012-12-15
• [Presentation] Rhodococcus erythropolis N771 由来一酸化窒素合成酵素及び酸化還元酵素様タンパク質の発現と生化学的解析 (2012)
  • Author(s): 北田亜美、前田悠貴、清水敏史、山中保明、大滝証、金城大進、山田和弘、野口恵一、養王田正文、尾高雅文
  • Organizer: 第85回日本生化学会大会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 2012-12-15
• [Presentation] Christal Structure Analysis of the Reaction Intermediate of Nitrile Hyd ratase (2012)
  • Author(s): Masafumi Odaka, Yasuaki, Yamanaka、Koichi Hashimoto, Keiichi Noguchi, Masafumi Yohda
  • Organizer: The 6th Asian Biological Inorganic Chemistry Conference
  • Place of Presentation: The Hong Kong Polytechnic University、香港
  • Year and Date: 2012-11-05
• [Presentation] Functions of Two Strictly Conserved Arginine Residues in Fe-type Hydratase from Rhodococcus erythropolis N771 (2012)
  • Author(s): Takayuki Aihara, Yasuaki Yamanaka, Keiichi Noguchi, Masafumi Yohda, Masafumi, Odaka
  • Organizer: 生命医薬情報学連合大会
  • Place of Presentation: Tower Hall Funabori、東京都
  • Year and Date: 2012-10-14
• [Presentation] 変異体を用いたチオシアネート加水分解酵素反応機構の解析 (2012)
  • Author(s): 渡邉俊則、山中保明、荒川孝俊.浪間聡志、中山洋、片山葉子、堂前直、野口恵一、養王田正文、尾高雅文
  • Organizer: 酵素工学研究会第68回講演会
  • Place of Presentation: 東京大学山上会館
  • Year and Date: 2012-10-05
• [Presentation] Rhodococcus erythropolis N771 由来一酸化窒素合成酵素及び酸化還元酵素様蛋白質の発現と生化学的解釈 (2012)
  • Author(s): 北田亜美、前田悠貴、清水聡史、山中保明、大滝証、金城大進、山田和弘、野口恵一、養王田正文、尾高雅文
  • Organizer: 酵素工学研究会第68回講演会
  • Place of Presentation: 東京大学山上会館
  • Year and Date: 2012-10-05
• [Presentation] ニトリルヒドラターゼファミリーの基質認識機構の違い (2012)
  • Author(s): 相原敬之、佐藤雅、山中保明、渡邉俊則、野口恵一、養王田正文、尾高雅文
  • Organizer: 酵素工学研究会第68回講演会
  • Place of Presentation: 東京大学山上会館
  • Year and Date: 2012-10-05
• [Presentation] ニトリルヒドラターゼ触媒機構の時間分割X線結晶構造解析 (2012)
  • Author(s): 山中保明、野口恵一、養王田正文、尾高雅文
  • Organizer: 第6回バイオ関連化学シンポジウム
  • Place of Presentation: 北海道大学
  • Year and Date: 2012-09-08
• [Presentation] チオシアネート加水分解酵素の成熟化機構 (2012)
  • Author(s): 中西拓也、堀祥太、荒川孝俊、中山洋、片山葉子、堂前直、養王田正文、尾高雅文
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場(招待講演)
  • Year and Date: 2012-06-22
• [Presentation] チオシアネート加水分解酵素基質ポケット近傍アルギニン残基の基質特異性への影響 (2012)
  • Author(s): 渡邉俊則、山中保明、荒川孝俊、浪間聡志、中山洋、片山葉子、堂前直、野口恵一、養王田正文、尾高雅文
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2012-06-20
• [Presentation] Corynebacterium sp. N-1074由来Halohydrin Hydrogen-halide-lyaseの構造解析 (2012)
  • Author(s): 渡辺文昭、大滝証、湯不二夫、尾高雄文、養王田正文
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2012-06-20
• [Presentation] Rhodococcus erythropolis N771 由来一酸化窒素合成酵素及び酸化還元酵素様タンパク質の発現と生化学的解釈 (2012)
  • Author(s): 北田亜美、前田悠樹、清水敏史、山中保明、大滝証、金城大進、山田和弘、野口恵一、養王田正文、尾高雅文
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2012-06-20
• [Presentation] Difference in the substrate recognition among nitrile hydratase family (2012)
  • Author(s): Takayuki Aihara, Masa Sato, Yasuaki Yamanaka, Toshinori Watanabe, Keiichi Noguchi, Masafumi Yohda, Masafumi Odaka
  • Organizer: 12th Japan - China-Korea Joint Symposium on Enzyme Enginnerin
  • Place of Presentation: Kanazwa Excel Hotel Tokyu, 石川県
  • Year and Date: 2012-05-29
• [Presentation] ニトリルヒドラターゼ触媒機構の時間分割結晶構造解析
  • Author(s): 山中保明、橋本浩一、野口恵一、養王田正文、尾高雅文
  • Organizer: 第13回 日本蛋白質科学会年会
  • Place of Presentation: 鳥取市、日本、とりぎん文化会館
• [Presentation] Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase
  • Author(s): M.Odaka､Y.Yamanaka、T.Watanabe、M.Yohda
  • Organizer: ICBIC16, Grenoble in 2013
  • Place of Presentation: Grenobres, France,Alpes Congres
• [Presentation] ニトリルヒドラターゼ触媒反応の時間分割X線結晶構造解析
  • Author(s): 山中保明、橋本浩一、野口恵一、養王田正文、尾高雅文
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: 横浜、日本、パシフィコ横浜
• [Presentation] TWO ARGININE RESIDUES IN THE SUBSTRATE POCKET PREDOMINANTLY CONTROL THE SUBSTRATE SELECTIVITY OF THIOCYANATE HYDROLASE
  • Author(s): Masafumi ODAKA､Yasuaki Yamanaka、Takatoshi Arakawa、Satoshi Namima、Shota Hori、Akashi Ohtaki、Keiichi Noguchi、Yoko Katayama、Masafumi Yohda
  • Organizer: Enzyme Engineering XXII
  • Place of Presentation: Toyama, Japan、富山国際会議場
• [Presentation] 時間分割Ｘ線結晶構造解析によるニトリルヒドラターゼ触媒反応機構の解明
  • Author(s): 山中保明、野口恵一、養王田正文、尾高雅文
  • Organizer: 錯体化学会 第63回討論会
  • Place of Presentation: 沖縄県、日本、琉球大学
• [Presentation] REACTION MECHANISM OF NITRILE HYDRATASE PROPOSED BY TIME RESOLVED X-RAY CRYSTALLOGRAPHY
  • Author(s): Yasuaki Yamanaka、Koichi Hashimoto、Keiichi Noguchi、Masafumi Yohda、Masafumi Odaka
  • Organizer: 54th Annual Conference of Association of Microbiologists of India
  • Place of Presentation: Haryana, INDIA、Maharshi Dayanand University