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Enzymological characterization of novel L-amino acid dehydrogenase for development of high specific agrochemicals

Research Project

Project/Area Number 16K18689
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeMulti-year Fund
Research Field Applied biochemistry
Research InstitutionTokai University

Principal Investigator

YONEDA Kazunari  東海大学, 農学部, 准教授 (00469397)

Project Period (FY) 2016-04-01 – 2018-03-31
Project Status Completed (Fiscal Year 2017)
Budget Amount *help
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2016: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
KeywordsL-スレオニン酸脱水素酵素 / 酵素化学 / 結晶構造解析 / 植物疫病菌 / L-スレオニン脱水素酵素 / NAD(P) / 選択性農薬 / X線結晶構造解析
Outline of Final Research Achievements

A gene encoding an L-Threonine dehydrogenase (ThrDH; PITG_05140) homologue was identified in the Phytophthora infestans. After transforming E. coli with pET-PITG_05140, an expression vector harboring the gene, the transformant cells exhibited a high level of L-ThrDH activity, and the enzyme was readily purified from the cells’ crude extract in one simple step: Talon metal affinity chromatography. The purified enzyme showed a single protein band on SDS-PAGE, and its N-terminal sequence was determined to be SDKIIHLTDD. SDS-PAGE showed the subunit molecular mass of P. infestans ThrDH to be about 55 kDa, which is consistent with the molecular weight calculated from the amino acid sequence. Non-tagged P. infestans ThrDH acted on L-threonine and DL-3-hydroxynorvaline, and the enzyme was partially inhibited by L-cysteine and N-acetylglycine.

Report

(3 results)
  • 2017 Annual Research Report   Final Research Report ( PDF )
  • 2016 Research-status Report
  • Research Products

    (24 results)

All 2018 2017 2016 Other

All Journal Article (8 results) (of which Peer Reviewed: 8 results,  Acknowledgement Compliant: 2 results) Presentation (13 results) (of which Int'l Joint Research: 1 results) Remarks (3 results)

  • [Journal Article] Crystal structure of the NADP and tartrate-bound complex of L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis2018

    • Author(s)
      Kazunari Yoneda, Haruhiko Sakuraba, Tomohiro Araki, Toshihisa Ohshima
    • Journal Title

      Extremophiles

      Volume: 22 Issue: 3 Pages: 395-405

    • DOI

      10.1007/s00792-018-1004-0

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Gene cloning and preliminary enzymological characterization of a gene homologous to NAD+-dependent L-aspartate dehydrogenase.2018

    • Author(s)
      Yoneda K, Araki T.
    • Journal Title

      Proceedings of School of Agriculture Tokai University.

      Volume: 37 Pages: 9-15

    • NAID

      120006456016

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri2017

    • Author(s)
      Junji Hayashi, Yuta Mutaguchi, Yume Minemura, Noriko Nakagawa, Kazunari Yoneda, Taketo Ohmori, Toshihisa Ohshima, Haruhiko Sakuraba
    • Journal Title

      Acta Crystallographica section D

      Volume: 73 Issue: 5 Pages: 428-437

    • DOI

      10.1107/s2059798317005332

    • NAID

      130007628792

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Structure-Based Engineering of an Artificially Generated NADP+-Dependent D-Amino Acid Dehydrogenase2017

    • Author(s)
      Junji Hayashi, Tomonari Seto, Hironaga Akita, Masahiro Watanabe, Tamotsu Hoshino, Kazunari Yoneda, Toshihisa Ohshima, Haruhiko Sakuraba
    • Journal Title

      Applied and Environmental Microbiology

      Volume: - Issue: 11

    • DOI

      10.1128/aem.00491-17

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Complete Amino Acid Sequence of a Copper/Zinc-Superoxide Dismutase from Ginger Rhizome.2017

    • Author(s)
      Nishiyama Y, Fukamizo T, Yoneda K, Araki T.
    • Journal Title

      Protein J.

      Volume: 36 Issue: 2 Pages: 98-107

    • DOI

      10.1007/s10930-017-9700-7

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Crystal structure analysis of extremely thermostable carbonyl reductase and structural basis for substrate recognition2017

    • Author(s)
      K.Yoneda, Y. Fukuda, T. Araki
    • Journal Title

      Proceedings of School of Agriculture Tokai University

      Volume: VOL. 36 Pages: 1-6

    • NAID

      40021168037

    • Related Report
      2016 Research-status Report
    • Peer Reviewed
  • [Journal Article] Catalytic properties and crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from the hyperthermophilic archaeon Aeropyrum pernix K12016

    • Author(s)
      Y. Fukuda, H. Sakuraba, T. Araki, T. Ohshima, K.Yoneda
    • Journal Title

      Enzyme and Microbial Technology

      Volume: VOL. 91 Pages: 17-25

    • DOI

      10.1016/j.enzmictec.2016.05.008

    • Related Report
      2016 Research-status Report
    • Peer Reviewed
  • [Journal Article] Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis2016

    • Author(s)
      Junji Hayashi, Kaori Yamamoto, Kazunari Yoneda, Toshihisa Ohshima, Haruhiko Sakuraba
    • Journal Title

      Proteins

      Volume: 84 Issue: 12 Pages: 1786-1796

    • DOI

      10.1002/prot.25161

    • Related Report
      2016 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Presentation] 植物疫病菌由来NAD(P)+依存性L-スレオニン脱水素酵素の機能・構造解析2018

    • Author(s)
      米田 一成、櫻庭 春彦、荒木 朋洋、大島 敏久
    • Organizer
      日本農芸化学会2018年度大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Geobacillus kaustophilus由来ジアミノピメリン酸脱炭酸酵素の機能解析2018

    • Author(s)
      大森 勇門、張 世文、米田 一成、大島 敏久
    • Organizer
      日本農芸化学会2018年度大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 超好熱アーキア由来FAD依存性L-プロリン脱水素酵素の構造解析2018

    • Author(s)
      大志田 達也、冨家 英嗣、川上 竜巳、里村 武範、米田 一成、大島 敏久、櫻庭 春彦
    • Organizer
      日本農芸化学会2018年度大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 超好熱アーキアPyrobaculum aerophilum由来galactose-1-phosphate uridylyltransferaseの構造解析2017

    • Author(s)
      花川大地、林順司、米田一成、大島敏久、櫻庭春彦
    • Organizer
      日本農芸化学会2017年度大会
    • Place of Presentation
      京都女子大学(京都府、京都市)
    • Year and Date
      2017-03-19
    • Related Report
      2016 Research-status Report
  • [Presentation] 超好熱性細菌Thermotoga maritima由来アスパラギン酸キナーゼ/ホモセリンデヒドロゲナーゼ融合酵素(AK/HseDH)の機能解析2017

    • Author(s)
      古場康平、林順司、安部展洋、米田一成、大島敏久、櫻庭春彦
    • Organizer
      日本農芸化学会2017年度大会
    • Place of Presentation
      京都女子大学(京都府、京都市)
    • Year and Date
      2017-03-19
    • Related Report
      2016 Research-status Report
  • [Presentation] ニワトリリゾチームおよびハマグリリゾチームの構造と機能に関する研究2017

    • Author(s)
      冨所 修平、米田 一成、荒木 朋洋
    • Organizer
      2017年度生命科学系学会合同年次大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 超好熱性細菌Thermotoga maritima由来アスパラギン酸キナーゼ/ホモセリンデヒドロゲナーゼ融合酵素(AK/HseDH)の分子特性2017

    • Author(s)
      古場 康平、林 順司、大志田 達也、安部 展洋、米田 一成、大島 敏久、櫻庭 春彦
    • Organizer
      日本農芸化学会 関西・中四国・西日本支部 2017年度合同大阪大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 超好熱アーキアPyrobaculum calidifontis由来グリセロール 1-リン酸脱水素酵素に見出した新規補酵素結合様式2017

    • Author(s)
      林 順司、山本 香李、米田 一成、大島 敏久、櫻庭 春彦
    • Organizer
      日本農芸化学会 関西・中四国・西日本支部 2017年度合同大阪大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 植物疫病菌由来NAD依存性L-スレオニン脱水素酵素の機能解析2017

    • Author(s)
      米田 一成、三神 卓也、櫻庭 春彦、荒木 朋洋、大島 敏久
    • Organizer
      日本農芸化学会 関西・中四国・西日本支部 2017年度合同大阪大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] ニワトリ脂肪肝由来NAD(P)H依存性カルボニル還元酵素の機能改変2017

    • Author(s)
      米田 一成、福田 雄大、櫻庭 春彦、荒木 朋洋、大島 敏久
    • Organizer
      日本ビタミン学会第69回大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 新規分岐鎖アミノ酸ラセマーゼ、イソロイシン2-エピメラーゼの構造解析2017

    • Author(s)
      林 順司、峰村 結芽、牟田口 祐太、米田 一成、大森 勇門、大島 敏久、櫻庭 春彦
    • Organizer
      日本ビタミン学会第69回大会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Structure and functional analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized(Tx) chicken fatty liver2016

    • Author(s)
      福田雄大、櫻庭春彦、荒木朋洋、大島敏久、米田一成
    • Organizer
      The First International Conference on Hybridized Agriculture
    • Place of Presentation
      崇城大学(熊本県、熊本市)
    • Year and Date
      2016-10-22
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research
  • [Presentation] 超好熱ア-キア由来NAD(P)H依存性カルボニル還元酵素の分子特性2016

    • Author(s)
      米田一成、福田雄大、櫻庭春彦、荒木朋洋、大島敏久
    • Organizer
      日本ビタミン学会第68回大会
    • Place of Presentation
      富山国際会議場(富山県、富山市)
    • Year and Date
      2016-06-17
    • Related Report
      2016 Research-status Report
  • [Remarks] 東海大学 農学部 バイオサイエンス学科 生物化学研究室 ホームページ

    • URL

      http://www2.kuma.u-tokai.ac.jp/~nougaku/Bio/yoneda/yoneda.htm

    • Related Report
      2017 Annual Research Report 2016 Research-status Report
  • [Remarks] 東海大学 農学部 バイオサイエンス学科 ホームページ

    • URL

      http://www2.kuma.u-tokai.ac.jp/~nougaku/Bio/biosci.htm

    • Related Report
      2017 Annual Research Report
  • [Remarks] 東海大学紀要 農学部 ホームページ

    • URL

      http://www2.kuma.u-tokai.ac.jp/kiyounougaku/

    • Related Report
      2017 Annual Research Report

URL: 

Published: 2016-04-21   Modified: 2019-03-29  

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