Elucidation of mechanisms for catalytic center maturation and catalysis of nitrile hydratase family enzyme

2011 Fiscal Year Final Research Report

• Project/Area Number: 21350089
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Chemistry related to living body
• Research Institution: Tokyo University of Agriculture and Technology
• Principal Investigator: ODAKA Masafumi 東京農工大学, 大学院・工学研究院, 准教授 (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)
• Project Period (FY): 2009 – 2011
• Keywords: 水和反応 / 加水分解 / 酵素反応 / 結晶構造解析 / 時間分割構造解析 / 翻訳後修飾 / タンパク質成熟化 / アクセサリータンパク質

Research Abstract

By performing time-resolved X-ray crystallography of Fe-type nitrile hydratase(NHase) whoseβArg56 were substituted by Lys, we obtained the structure of the enzyme-substrate complex. By replacing two Arg residues resided in the active site pocket of a NHase family enzyme, thiocyanate hydrolase(SCNase), we showed that the size as well as the surface charge of the substrate binding pocket was important for the substrate specificities of SCNase. As for the maturation mechanism, we found that the SCNase activator protein, P15K, is responsible for the refolding, metal-insertion and post-translational modification of Cys ligand in the catalytic subunit,γ. The metal-incorporatedγsubunit form the complex with the other subunits,αandβ, to form the mature SCNase.

Research Products

• [Journal Article] Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase (2010)
  • Author(s): Yamanaka Y., Hashimoto K., Ohtaki A., Noguchi K., Yohda M., and Odaka M.
  • Journal Title: J. Biol. Inorg. Chem Volume: 15 Pages: 655-665
  • Peer Reviewed
• [Journal Article] Structure and characterization of amidase from Rhodococcus sp. N-771 : Insight into the molecular mechanism of substrate recognition (2010)
  • Author(s): Ohtaki A., Murata K., Sato Y., Noguchi K., Miyatake H., Dohmae N., Yamada K., Yohda M., and Odaka M.
  • Journal Title: Biochim. Biophys. Acta Volume: 1804 Pages: 184-192
  • Peer Reviewed
• [Journal Article] Structural basis for catalytic activation of thiocyanate hydrolase involving metal-ligated cysteine modification (2009)
  • Author(s): Arakawa T., Kawano Y., Katayama Y., Nakayama H., Dohmae N., Yohda M., and Odaka M
  • Journal Title: J. Am. Chem. Soc Volume: 131 Pages: 14838-14843
  • Peer Reviewed
• [Presentation] Effect of arginine residues around the substrate binding pocket on the substrate specificity of thiocyanate hydrolase (2011)
  • Author(s): YamanakaY., Arakawa T., Namima S., Hori S., Ohtaki A., Noguchi N., Katayama Y., Yohda M., and Odaka M.
  • Organizer: 12th European Biological Inorganic Chemistry Conference
  • Place of Presentation: Vancouver, Canada
  • Year and Date: 2011-08-10
• [Presentation] Catalytic Mechanism of Nitrile Hydratase Proposed by Time-Resolved X-Ray Crystallography (2011)
  • Author(s): Yohda M., Hashimoto K., and Odaka M.
  • Organizer: The 12th International Conference on QiR(Quality in Resarch)
  • Place of Presentation: Bali, Indonesia
  • Year and Date: 2011-07-05
• [Presentation] Biochemical and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in Nitril Hydratase (2010)
  • Author(s): Yamanaka Y., Hashimoto K., Ohtaki A., Noguchi K., Yohda M. and Odaka M.
  • Organizer: EUROBIC 10(10th European Biological Inorganic Chemistry Conference)
  • Place of Presentation: Thessaloniki, Greece
  • Year and Date: 2010-07-22
• [Presentation] Structural Basis for Catalytic Activation of Thiocyanate Hydrolase Involving Metal-Ligated Cysteine Modification (2010)
  • Author(s): Odaka M., Arakawa T., Kartayama Y., Nakayama H., Dohmae N., Yohda M.
  • Organizer: EUROBIC 10(10th European Biological Inorganic Chemistry Conference)
  • Place of Presentation: Thessaloniki, Greece
  • Year and Date: 2010-07-22
• [Presentation] アクリルアミド生産に使われる酵素ニトリルヒドラターゼの反応機構 (2010)
  • Author(s): 尾高雅文,橋本浩一,山中保明,大滝証,野口恵一,養王田正文
  • Organizer: 日本化学会第90年会ATPシンポジウム「物質・エネルギー変換材料」
  • Place of Presentation: 大阪
  • Year and Date: 2010-03-26
• [Presentation] アクリルアミド生産に使われる酵素ニトリルヒドラターゼの触媒機構解明の現状 (2010)
  • Author(s): 尾高雅文
  • Organizer: H21年度生命物質構造解析研究会
  • Place of Presentation: 東京
  • Year and Date: 2010-01-15
• [Presentation] Catalytic mechanism of nitrile hydratase proposed by time-resolved X-ray crystallography using a novel substrate, tert-butylisonitrile (2009)
  • Author(s): Odaka M., Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M.
  • Organizer: The 14th International Conference on Biological Inorganic Chemistry(ICBIC14)
  • Place of Presentation: Nagoya, Japan
  • Year and Date: 2009-07-30
• [Remarks]
  • URL: http://www.tuat.ac.jp/~yohda/index.html