Accurate detection of adenylation domain functions in nonribosomal peptide synthetases by an enzyme-linked immunosorbent assay system using active site-directed probes for adenylation domains

• Project/Area Number: 26750370
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Chemical biology
• Research Institution: Kyoto University
• Principal Investigator: Ishikawa Fumihiro 京都大学, 学内共同利用施設等, 助教 (50631553)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 非リボソーム性ペプチド / 非リボソーム性ペプチド合成酵素 / アデニレーションドメイン / 化学プローブ / ELISA / 酵素機能改変 / 合成分子プローブ

Outline of Final Research Achievements

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we developed an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality
that immobilizes probe molecules onto a streptavidin-coated solid support.When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes.

Research Products

• [Journal Article] Affinity purification method for the identification of nonribosomal peptide biosynthetic enzymes using a synthetic probe for adenylation domains (2016)
  • Author(s): Fumihiro Ishikawa and Hideaki Kakeya
  • Journal Title: Methods Mol. Biol. Volume: 1401 Pages: 63-76
  • DOI: 10.1007/978-1-4939-3375-4_4
  • ISBN: 9781493933730, 9781493933754
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A competitive enzyme-linked immunosorbent assay system for adenylation domains in nonribosomal peptide synthetases (2016)
  • Author(s): Fumihiro Ishikawa and Hideaki Kakeya
  • Journal Title: ChemBioChem Volume: 17 Issue: 6 Pages: 474-478
  • DOI: 10.1002/cbic.201500553
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] 合成小分子化合物群を用いたペプチド性天然化合物合成酵素の選択的ラベル化技術の開発と応用 (2015)
  • Author(s): 石川文洋
  • Journal Title: 日本化学会生体機能関連化学部会NEWS LETTER Volume: 30 Pages: 2-5
  • Acknowledgement Compliant
• [Journal Article] Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains (2015)
  • Author(s): Fumihiro Ishikawa, Kengo Miyamoto, Sho Konno, Shota Kasai, Hideaki Kakeya
  • Journal Title: ACS Chemical Biology Volume: 10 Issue: 12 Pages: 2816-2826
  • DOI: 10.1021/acschembio.5b00595
  • Peer Reviewed
• [Journal Article] A multiple-labeling strategy for nonribosomal peptide synthetases using active-site-directed proteomic probes for adenylation domains (2015)
  • Author(s): Fumihiro Ishikawa, Takehiro Suzuki, Naoshi Dohmae, and Hideaki Kakeya
  • Journal Title: ChemBioChem Volume: 16 Issue: 18 Pages: 2590-2594
  • DOI: 10.1002/cbic.201500481
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Functional Profiling of Adenylation Domains in Nonribosomal Peptide Synthetases by Competitive Activity-based Protein Profiling (2015)
  • Author(s): Shota Kasai, Sho Konno, Fumihiro Ishikawa Hideaki Kakeya
  • Journal Title: Chemical Communications Volume: 51 Issue: 87 Pages: 15764-15767
  • DOI: 10.1039/c5cc04953a
  • Peer Reviewed / Open Access
• [Journal Article] Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains (2015)
  • Author(s): Fumihiro Ishikawa, Sho Konno, Takehiro Suzuki, Naoshi Dohmae, Hideaki Kakeya
  • Journal Title: ACS Chemical Biology Volume: 10 Issue: 9 Pages: 1989-1997
  • DOI: 10.1021/acschembio.5b00097
  • Peer Reviewed
• [Journal Article] Recent advances in adenylation domain enzymology in nonribosomal peptide biosynthesis (2015)
  • Author(s): Fumihiro Ishikawa and Hideaki Kakeya
  • Journal Title: Curr. Org. Chem. Volume: 19 Issue: 13 Pages: 1204-1221
  • DOI: 10.2174/1385272819666150410003958
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Affinity purification method for the identification of nonribosomal peptide biosynthetic enzymes using a synthetic probe for adenylation domains (2015)
  • Author(s): Fumihiro Ishikawa* and Hideaki Kakeya*
  • Journal Title: Methods Mol. Biol. Volume: XX
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases (2015)
  • Author(s): Sho Konno, Fumihiro Ishikawa,* Takehiro Suzuki, Naoshi Dohmae, Michael D. Burkart, and Hideaki Kakeya*
  • Journal Title: Chem. Commun. Volume: 51 Issue: 12 Pages: 2262-2265
  • DOI: 10.1039/c4cc09412c
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Biosynthetic origins of the epoxyquinone skeleton in epoxyquinols A and B (2014)
  • Author(s): Katsuki Fujita, Fumihiro Ishikawa, and Hideaki Kakeya
  • Journal Title: J. Nat. Prod. Volume: 77 Issue: 12 Pages: 2707-2710
  • DOI: 10.1021/np5004615
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] 合成小分子化合物群を用いたペプチド性天然化合物合成酵素の選択的ラベル化技術の開発と応用 (2015)
  • Author(s): 石川文洋
  • Organizer: 生理化学研究ユニット 第5回シンポジウム
  • Place of Presentation: 京都
  • Year and Date: 2015-12-25
• [Presentation] Accurate detection and manipulation of adenylation domain functions in nonribosomal peptide synthetases by an enzyme-linked immunosorbent assay system using active site-directed probes for adenylation domains (2015)
  • Author(s): Fumihiro Ishikawa, Kengo Miyamoto, Sho Konno, Shota Kasai, and Hideaki Kakeya
  • Organizer: The International Chemical Congress of Pacific Basin Societies 2015 (PACIFICHEM 2015)
  • Place of Presentation: Hawaii
  • Year and Date: 2015-12-15
  • Int'l Joint Research
• [Presentation] Profiling of nonribosomal peptide synthetase activities using active site-directed proteomic probes for adenylation domains (2015)
  • Author(s): Sho Konno, Fumihiro Ishikawa, Takehiro Suzuki, Naoshi Dohmae, and Hideaki Kakeya
  • Organizer: The International Chemical Congress of Pacific Basin Societies 2015 (PACIFICHEM 2015)
  • Place of Presentation: Hawaii
  • Year and Date: 2015-12-15
  • Int'l Joint Research
• [Presentation] 非リボソーム性ペプチド合成酵素の翻訳後修飾を標的とした化学プローブの開発 (2015)
  • Author(s): 笠井昭太, 石川文洋, 掛谷秀昭
  • Organizer: 第65回日本薬学会近畿支部総会
  • Place of Presentation: 大阪
  • Year and Date: 2015-10-17
• [Presentation] 合成小分子化合物群によるアデニレーションドメインの選択的標識化およびプロファイリングへの展開 (2015)
  • Author(s): 石川文洋, 今野翔, 笠井昭太, 鈴木健裕, 堂前直, 掛谷秀昭
  • Organizer: 第9回バイオ関連化学シンポジウム
  • Place of Presentation: 熊本
  • Year and Date: 2015-09-10
• [Presentation] 活性部位指向型プローブを利用したアデニレーションドメインの機能的プロファイリング (2015)
  • Author(s): 今野翔, 笠井昭太, 石川文洋, 掛谷秀昭
  • Organizer: 第9回バイオ関連化学シンポジウム
  • Place of Presentation: 熊本
  • Year and Date: 2015-09-10
• [Presentation] アデニレーションドメインに対する活性部位指向型プローブを用いた非リボソーム性ペプチド合成酵素活性のプロファイリング (2015)
  • Author(s): 今野翔, 石川文洋, 鈴木健裕, 堂前 直, 掛谷秀昭
  • Organizer: 第15回日本蛋白質科学会年会
  • Place of Presentation: 徳島
  • Year and Date: 2015-06-24
• [Presentation] 非リボソーム性ペプチド合成酵素の担体タンパク質を標的とした低分子プローブの開発 (2015)
  • Author(s): 笠井昭太, 石川文洋, 掛谷秀昭
  • Organizer: 日本ケミカルバイオロジー学会・第10回年会
  • Place of Presentation: 仙台
  • Year and Date: 2015-06-10
• [Presentation] アデニレーションドメインに対する活性部位指向型プローブを利用した非リボソーム性ペプチド合成酵素活性のプロファイリング (2015)
  • Author(s): 今野翔, 石川文洋, 鈴木健裕, 堂前直, 掛谷秀昭
  • Organizer: 日本ケミカルバイオロジー学会・第10回年会
  • Place of Presentation: 仙台
  • Year and Date: 2015-06-10
• [Presentation] アデニレーションドメインに対する活性部位指向型プローブの開発 (2015)
  • Author(s): 今野翔、石川文洋、鈴木健裕、堂前直、掛谷秀昭
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸
  • Year and Date: 2015-03-25
• [Presentation] 非リボソーム性ペプチド合成酵素のぺプチジル担体タンパク質を標的としたプローブの開発研究 (2015)
  • Author(s): 笠井昭太, 石川文洋, 掛谷秀昭
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸
  • Year and Date: 2015-03-25
• [Presentation] Chemical Biology for modulators targeting cancer microenvironment and cell membrane signaling. (2015)
  • Author(s): Kakeya, H., Yoshimura, A., Sugiyama, R., Kishimoto, S., Nishimura, S., Hattori, A., Ishikawa, F., Yomoda, Y., Lu, S., Katagiri, N., Shimada, S., Seto, E., Hayashi, H., Takahashi, N., Harada, H.
  • Organizer: Natural Products 2015; Natural product discovery & development in the post genomic era
  • Place of Presentation: San Diego
  • Year and Date: 2015-01-11
• [Presentation] 活性部位指向型プローブはアデニレーションドメインを高選択的に標識する. 第17回生命化学研究会, 高知, 1月, 2015. (2015)
  • Author(s): 石川文洋, 今野翔, 掛谷秀昭
  • Organizer: 第17回生命化学研究会
  • Place of Presentation: 高知
  • Year and Date: 2015-01-08
• [Presentation] アデニレーションドメインに対するテーラーメイドケミカルラベリング法の開発 (2014)
  • Author(s): 石川文洋, 今野翔, 掛谷秀昭
  • Organizer: 第20回天然薬物の開発と応用シンポジウム
  • Place of Presentation: 東京
  • Year and Date: 2014-11-05
• [Presentation] 活性部位指向型プローブを用いたアデニレーションドメインの選択的標識化 (2014)
  • Author(s): 今野翔, 石川文洋, 掛谷秀昭
  • Organizer: 第9回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-11
• [Presentation] 進化分子工学を指向したアデニレーションドメインに対するELISA法の開発 (2014)
  • Author(s): 石川 文洋, 宮本 健吾, 今野 翔, 笠井 昭太, 掛谷 秀昭
  • Organizer: 第9回バイオ関連化学シンポジウム
  • Place of Presentation: 岡山
  • Year and Date: 2014-09-11
• [Presentation] Chemical labeling of adenylation domains in nonribosomal peptide synthetase (2014)
  • Author(s): Konno, S., Ishikawa, S., Kakeya, H
  • Organizer: Bioorganic Chemistry-Global Challenges in the 21st Century-
  • Place of Presentation: Andover
  • Year and Date: 2014-06-08
• [Presentation] Highly selective detection method for nonribosomal peptide synthetase using cocktails of active-site probes for adenylation domains (2014)
  • Author(s): Ishikawa, F., Konno, S., Kakeya, H
  • Organizer: Gordon Research Conferences: Bioorganic Chemistry-Global Challenges in the 21st Century-
  • Place of Presentation: Andover
  • Year and Date: 2014-06-08
• [Presentation] Chemical labeling of adenylation domains in nonribosomal peptide synthetases (2014)
  • Author(s): Fumihiro Ishikawa
  • Organizer: host: Sheryl Tsai
  • Place of Presentation: UC Irvine
  • Year and Date: 2014-06-06
  • Invited
• [Remarks] 京都大学大学院 薬学研究科 医薬創成情報科学専攻 システムケモセラピー・制御分子学分野
  • URL: http://www.pharm.kyoto-u.ac.jp//sc-molsci/