Updated on 2024/07/13

写真a

 
SATO Tsutomu
 
Organization
Academic Assembly Institute of Science and Technology NOUGAKU KEIRETSU Professor
Faculty of Agriculture Department of Agriculture Professor
Title
Professor
External link

Degree

  • 農学博士 ( 2000.3   新潟大学 )

Research Interests

  • 生合成

  • 香料

  • イソプレノイド

  • テルペノイド

  • 天然物

  • テルペン

Research Areas

  • Life Science / Bioorganic chemistry  / 生合成

Research History (researchmap)

  • Niigata University   Professor

    2020.5

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  • Niigata University   Faculty of Agriculture

    2019.12 - 2020.4

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  • Niigata University   Associate Professor

    2007.4

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  • Niigata University   Associate Professor (as old post name)

    2004.10 - 2007.3

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  • Niigata University   Faculty of Agriculture   Research Assistant

    2001.9 - 2004.9

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  • Niigata University   Faculty of Agriculture

    2000.4 - 2001.8

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Research History

  • Niigata University   Faculty of Agriculture Department of Agriculture   Professor

    2020.5

  • Niigata University   Faculty of Agriculture Department of Agriculture   Associate Professor

    2017.4 - 2020.5

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Associate Professor

    2004.9 - 2020.5

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Associate Professor

    2004.9 - 2020.5

  • Niigata University   Faculty of Agriculture Department of Applied Biological Chemistry   Associate Professor

    2004.9 - 2017.3

Education

  • 新潟大学博士後期課程修了

    2000.3

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Professional Memberships

  • JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY

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Committee Memberships

  • 日本農芸化学会   評議員  

    2016.4   

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    Committee type:Academic society

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Papers

  • Identification and functional analysis of a new type of Z,E-mixed prenyl reductase from mycobacteria. International journal

    Tohru Abe, Mariko Hakamata, Akihito Nishiyama, Yoshitaka Tateishi, Sohkichi Matsumoto, Hisashi Hemmi, Daijiro Ueda, Tsutomu Sato

    The FEBS journal   289 ( 16 )   4981 - 4997   2022.2

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Isoprenoids with reduced Z,E-mixed prenyl groups are found in various organisms. To date, only polyprenol reductases (PR-Dol) involved in dolichol biosynthesis have been identified as enzymes capable of reducing Z,E-mixed prenyl groups. Although C35 -isoprenoids with reduced Z,E-mixed prenyl groups are found in mycobacteria, Z,E-mixed heptaprenyl reductase (HepR) remains unidentified. In the present study, the identification and functional analysis of HepR was performed. No PR-Dol homolog gene was detected in the genome of Mycolicibacterium vanbaalenii. However, a homolog of geranylgeranyl reductase (GGR), which reacts with an all-E prenyl group as a substrate, was encoded in the genome; thus, we analyzed it as a HepR candidate. In vitro enzymatic assay and in vivo gene suppression analysis identified the GGR homolog as HepR and revealed that HepR catalyzes the reduction of ω- and E- prenyl units in Z,E-mixed heptaprenyl diphosphates, and C35 -isoprenoids are mainly biosynthesized using E,E,E-geranylgeranyl diphosphate as a precursor. Thus, it was demonstrated that the Z,E-mixed prenyl reductase family exists in the GGR homologs. To the best of our knowledge, this is the first identification of a new type of Z,E-mixed prenyl reductase with no sequence homology to PR-Dol. The substrate specificity of HepR significantly differed from that of GGR, suggesting that it is a new enzyme. HepR homologs are widely distributed in mycobacterial genomes, and lipid analysis suggests that many strains, including pathogenic species, produce HepR metabolites. The discovery of this new enzyme will promote further research on Z,E-mixed isoprenoids.

    DOI: 10.1111/febs.16412

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  • Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein. Reviewed International journal

    Yota Yamabe, Yukina Kawagoe, Kotone Okuno, Mao Inoue, Kanako Chikaoka, Daijiro Ueda, Yuko Tajima, Tadasu K Yamada, Yoshito Kakihara, Takashi Hara, Tsutomu Sato

    Scientific reports   10 ( 1 )   19643 - 19643   2020.11

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    Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, "ambrein synthase," for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8-15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid β-induced apoptosis, were discovered using the synthesized ambrein.

    DOI: 10.1038/s41598-020-76624-y

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  • Characterization of Class IB Terpene Synthase: The First Crystal Structure Bound with a Substrate Surrogate Reviewed

    Rafaella Stepanova, Hayato Inagi, Kei Sugawara, Kazuya Asada, Tomoyuki Nishi, Daijiro Ueda, Yoko Yasuno, Tetsuro Shinada, Kunio Miki, Masahiro Fujihashi, Tsutomu Sato

    ACS Chemical Biology   15 ( 6 )   1517 - 1525   2020.6

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    Authorship:Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/acschembio.0c00145

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  • Insight into Isoprenoid Biosynthesis by Functional Analysis of Isoprenyl Diphosphate Synthases from Mycobacterium vanbaalenii and Mycobacterium tuberculosis. Reviewed International journal

    Tohru Abe, Sadamu Ozaki, Daijiro Ueda, Tsutomu Sato

    Chembiochem : a European journal of chemical biology   21   2931 - 2938   2020.6

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Comprehensive functional analyses of E-isoprenyl diphosphate synthases (E-IDSs) from nonpathogenic Mycobacterium vanbaalenii have been performed. Mv0992 and Mv1577 represent a nonaprenyl diphosphate (E-C45 ) synthase and a geranylgeranyl diphosphate (E-C20 ) synthase, respectively. Although Mv3536 was identified as an E-C20 synthase using a single enzyme, co-incubation of Mv3536 and Z-IDSs (Mv4662 and Mv3822) strongly suggested it releases an intermediate geranyl diphosphate (E-C10 ) during a continuous condensation reaction. Mv0992 and Mv3536 functions differed from those of the previously reported pathogenic Mycobacterium tuberculosis homologues Rv0562 and Rv2173, respectively. Re-analysis of Rv0562 and Rv2173 demonstrated that their functions were similar to those of Mv0992 and Mv3536 (Rv0562: E-C45 synthase; Rv2173: E-C10-15 synthase). The newly proposed functions of Rv0562 and Rv2173 would be in the biosynthesis of menaquinone and glycosyl carrier lipids essential for growth. Furthermore, a reduced allylic diphosphate could be used as the Z-IDS of the Mv3822 substrate, thereby introducing a potentially novel pathway of cyclic sesquarterpene biosynthesis.

    DOI: 10.1002/cbic.202000235

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  • A Non-Enzymatic Pathway with Superoxide in Intracellular Terpenoid Synthesis. Reviewed International journal

    Daijiro Ueda, Saori Matsugane, Wataru Okamoto, Masayuki Hashimoto, Tsutomu Sato

    Angewandte Chemie (International ed. in English)   57 ( 32 )   10347 - 10351   2018.8

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Non-C5 -units terpenoids (norisoprenoids) with an acetonyl group are widely distributed in nature. However, studies on the biosynthesis of norisoprenoids are scarce. Now, the C33 norisoprenoid, (all-E)-farnesylfarnesylacetone, was identified from Bacillus spp. and it was elucidated for the first time that superoxide mediates the cleavage of menaquinones (vitamin K) to form norisoprenoids in saponification treatment. From in vivo experiments using gene-disrupted Bacillus subtilis strains targeted for enzymes responsible for menaquinone biosynthesis and for superoxide dismutase, it was suggested that the non-enzymatic cleavage (autoxidation) of menaquinone with superoxide resulted in norisoprenoid synthesis in Bacillus cells. Furthermore, the bioactive norisoprenoids, farnesylacetone and phytone, were produced in Bacillus cells by this novel synthesis system.

    DOI: 10.1002/anie.201805383

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  • Crystal structure and functional analysis of large-terpene synthases belonging to a newly found subclass. Reviewed International journal

    Masahiro Fujihashi, Tsutomu Sato, Yuma Tanaka, Daisuke Yamamoto, Tomoyuki Nishi, Daijiro Ueda, Mizuki Murakami, Yoko Yasuno, Ai Sekihara, Kazuma Fuku, Tetsuro Shinada, Kunio Miki

    Chemical science   9 ( 15 )   3754 - 3758   2018.4

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Thousands of terpenes have been identified to date. However, only two classes of enzymes are known to be involved in their biosynthesis, and each class has characteristic amino-acid motifs. We recently identified a novel large-terpene (C25/C30/C35) synthase, which shares no motifs with known enzymes. To elucidate the molecular mechanism of this enzyme, we determined the crystal structure of a large-β-prene synthase from B. alcalophilus (BalTS). Surprisingly, the overall structure of BalTS is similar to that of the α-domain of class I terpene synthases although their primary structures are totally different from each other. Two novel aspartate-rich motifs, DYLDNLxD and DY(F,L,W)IDxxED, are identified, and mutations of any one of the aspartates eliminate its enzymatic activity. The present work leads us to propose a new subclass of terpene synthases, class IB, which is probably responsible for large-terpene biosynthesis.

    DOI: 10.1039/c8sc00289d

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  • Cyclization of Squalene from Both Termini: Identification of an Onoceroid Synthase and Enzymatic Synthesis of Ambrein Reviewed

    Daijiro Ueda, Tsutomu Hoshino, Tsutomu Sato

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   135 ( 49 )   18335 - 18338   2013.12

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER CHEMICAL SOC  

    The onoceroids are triterpenoids biosynthesized from squalene or (3S)-2,3-oxidosqualene by cyclization from both termini. We recently revealed that tetraprenyl-beta-curcumene cyclase from Bacillus megaterium (BmeTC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-beta-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. Here, we reveal that BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase. We also report the first onoceroids from bacterial origin. Our discoveries suggest that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of BmeTC enabled the synthesis of (+)-ambrein, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase-catalyzed reaction from easily available squalene.

    DOI: 10.1021/ja4107226

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  • Bifunctional Triterpene/Sesquarterpene Cyclase: Tetraprenyl-beta-curcumene Cyclase Is Also Squalene Cyclase in Bacillus megaterium Reviewed

    Tsutomu Sato, Hiroko Hoshino, Satoru Yoshida, Mami Nakajima, Tsutomu Hoshino

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   133 ( 44 )   17540 - 17543   2011.11

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER CHEMICAL SOC  

    This study demonstrates that a tetraprenyl-beta-curcumene cyclase, which was originally identified as a sesquarterpene cyclase that converts a head-to-tail type of monocycle to a pentacycle, also cyclizes a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8 alpha-hydroxypolypoda-13,17,21-triene. The 8 alpha-hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium. It was also demonstrated that cyclizations of both tetraprenyl-beta-curcumene and squalene occurred with a purified B. megaterium TC homologue in the same reaction mixture. These results suggest that the tetraprenyl-beta-curcumene cyclase is bifunctional, cyclizing both tetraprenyl-beta-curcumene and squalene in vivo. This is the first report describing a bifunctional terpene cyclase, which biosynthesizes two classes of cyclic terpenes with different numbers of carbons as natural products in the organism.

    DOI: 10.1021/ja2060319

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  • Sesquarterpenes (C-35 Terpenes) Biosynthesized via the Cyclization of a Linear C-35 Isoprenoid by a Tetraprenyl-beta-curcumene Synthase and a Tetraprenyl-beta-curcumene Cyclase: Identification of a New Terpene Cyclase Reviewed

    Tsutomu Sato, Satoru Yoshida, Hiroko Hoshino, Mizuki Tanno, Mami Nakajima, Tsutomu Hoshino

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   133 ( 25 )   9734 - 9737   2011.6

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    In this study, mono- and pentacyclic C-35 terpenes from Bacillus subtilis were biosynthesized via the cydization of C-35 isoprenoid using purified enzymes, including the first identified new terpene cydase that shows no sequence homology to any of the known terpene cyclases. On the basis of these findings, we propose that these C-35 terpenes should be called the new family of "sesquarterpenes."

    DOI: 10.1021/ja203779h

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  • Analysis of vitamin D receptor binding affinities of enzymatically synthesized triterpenes including ambrein and unnatural onoceroids. International journal

    Daijiro Ueda, Natsu Matsuda, Yuka Takaba, Nami Hirai, Mao Inoue, Taichi Kameya, Tohru Abe, Nao Tagaya, Yasuhiro Isogai, Yoshito Kakihara, Florian Bartels, Mathias Christmann, Tetsuro Shinada, Kaori Yasuda, Tsutomu Sato

    Scientific reports   14 ( 1 )   1419 - 1419   2024.1

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    Onoceroids are a rare family of triterpenes. One representative onoceroid is ambrein, which is the main component of ambergris used as a traditional medicine. We have previously identified the onoceroid synthase, BmeTC, in Bacillus megaterium and succeeded in creating ambrein synthase by introducing mutations into BmeTC. Owing to the structural similarity of ambrein to vitamin D, a molecule with diverse biological activities, we hypothesized that some of the activities of ambergris may be induced by the binding of ambrein to the vitamin D receptor (VDR). We demonstrated the VDR binding ability of ambrein. By comparing the structure-activity relationships of triterpenes with both the VDR affinity and osteoclastic differentiation-promoting activity, we observed that the activity of ambrein was not induced via the VDR. Therefore, some of the activities of ambergris, but not all, can be attributed to its VDR interaction. Additionally, six unnatural onoceroids were synthesized using the BmeTC reactions, and these compounds exhibited higher VDR affinity than that of ambrein. Enzymatic syntheses of onoceroid libraries will be valuable in creating a variety of bioactive compounds beyond ambergris.

    DOI: 10.1038/s41598-024-52013-7

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  • Structural-model-based genome mining can efficiently discover novel non-canonical terpene synthases hidden in genomes of diverse species

    Tohru Abe, Haruna Shiratori, Kosuke Kashiwazaki, Kazuma Hiasa, Daijiro Ueda, Tohru Taniguchi, Hajime Sato, Takashi Abe, Tsutomu Sato

    Chemical Science   2024

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    Publishing type:Research paper (scientific journal)   Publisher:Royal Society of Chemistry (RSC)  

    Non-canonical terpene synthases with primary sequences that are unrecognizable as canonical terpene synthases could be discovered through structural-model-based genome mining.

    DOI: 10.1039/d4sc01381f

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  • Synthesis of Drimane-8α,11-diol Using Terpene Cyclase from <i>Bacillus megaterium</i>

    Keita Ozawa, Yuki Yamamoto, Eigo Fukuda, Seiya Endo, Atsushi Nakayama, Yoko Yasuno, Daijiro Ueda, Tsutomu Sato, Tetsuro Shinada

    Chemistry Letters   52 ( 6 )   520 - 523   2023.5

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    DOI: 10.1246/cl.230151

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  • Insight into the mechanism of geranyl-β-phellandrene formation catalyzed by Class IB terpene synthases. International journal

    Shogo Iwakata, Kazuya Asada, Tomoyuki Nishi, Rafaella Stepanova, So Shinoda, Daijiro Ueda, Masahiro Fujihashi, Yoko Yasuno, Tetsuro Shinada, Tsutomu Sato

    Bioscience, biotechnology, and biochemistry   86 ( 6 )   724 - 729   2022.5

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    Terpene synthase (TS) from Bacillus alcalophilus (BalTS) is the only Class IB TS for which a 3D structure has been elucidated. Recently, geranyl-β-phellandrene, a novel cyclic diterpene, was identified as a product of BalTS in addition to the acyclic β-springene. In the present study, we have provided insight into the mechanism of geranyl-β-phellandrene formation. Deuterium labeling experiments revealed that the compound is produced via a 1,3-hydride shift. In addition, nonenzymatic reactions using divalent metal ions were performed. The enzyme is essential for the geranyl-β-phellandrene formation. Furthermore, BalTS variants targeting tyrosine residues enhanced the yield of geranyl-β-phellandrene and the proportion of the compound of the total products. It was suggested that the expansion of the active site space may allow the conformation of the intermediates necessary for cyclization. The present study describes the first Class IB TSs to successfully alter product profiles while retaining high enzyme activity.

    DOI: 10.1093/bbb/zbac036

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  • Adduct Formation of Delamanid with NAD in Mycobacteria. Reviewed International journal

    Mikayo Hayashi, Akihito Nishiyama, Ryuki Kitamoto, Yoshitaka Tateishi, Mayuko Osada-Oka, Yukiko Nishiuchi, Shaban A Kaboso, Xiuhao Chen, Mamoru Fujiwara, Yusuke Inoue, Yoshikazu Kawano, Masanori Kawasaki, Tohru Abe, Tsutomu Sato, Kentaro Kaneko, Kimiko Itoh, Sohkichi Matsumoto, Makoto Matsumoto

    Antimicrobial agents and chemotherapy   64 ( 5 )   2020.4

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    Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F420)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (ndh) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.

    DOI: 10.1128/AAC.01755-19

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  • Adduct Formation of Delamanid with NAD in Mycobacteria

    Mikayo Hayashi, Akihito Nishiyama, Ryuki Kitamoto, Yoshitaka Tateishi, Mayuko Osada-Oka, Yukiko Nishiuchi, Shaban A. Kaboso, Xiuhao Chen, Mamoru Fujiwara, Yusuke Inoue, Yoshikazu Kawano, Masanori Kawasaki, Tohru Abe, Tsutomu Sato, Kentaro Kaneko, Kimiko Itoh, Sohkichi Matsumoto, Makoto Matsumoto

    Antimicrobial Agents and Chemotherapy   64 ( 5 )   2020.4

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    Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F
    <sub>420</sub>
    )-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated
    <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
    var.

    DOI: 10.1128/aac.01755-19

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  • Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases Reviewed

    Qingwen Chen, Jianxu Li, Zhixi Liu, Takaaki Mitsuhashi, Yuting Zhang, Haili Liu, Yihua Ma, Juan He, Tetsuro Shinada, Tsutomu Sato, Yong Wang, Hongwei Liu, Ikuro Abe, Peng Zhang, Guodong Wang

    Plant Communications   1 ( 5 )   100051 - 100051   2020.4

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    © 2020 The Author(s) Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana. AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications.

    DOI: 10.1016/j.xplc.2020.100051

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  • Japanese Rice Wine can reduce psychophysical stress-induced depression-like behaviors and Fos expression in the trigeminal subnucleus caudalis evoked by masseter muscle injury in the rats. Reviewed

    Nakatani Y, Kakihara Y, Shimizu S, Kurose M, Sato T, Kaneoke M, Saeki M, Takagi R, Yamamura K, Okamoto K

    Bioscience, biotechnology, and biochemistry   83 ( 1 )   1 - 11   2018.10

  • An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid. Reviewed International journal

    Haruna Saeki, Ryota Hara, Hironobu Takahashi, Miu Iijima, Ryosuke Munakata, Hiromichi Kenmoku, Kazuma Fuku, Ai Sekihara, Yoko Yasuno, Tetsuro Shinada, Daijiro Ueda, Tomoyuki Nishi, Tsutomu Sato, Yoshinori Asakawa, Fumiya Kurosaki, Kazufumi Yazaki, Futoshi Taura

    Plant physiology   178 ( 2 )   535 - 551   2018.10

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    Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.

    DOI: 10.1104/pp.18.00655

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  • Alicyclobacillus acidocaldarius Squalene-Hopene Cyclase: The Critical Role of Steric Bulk at Ala306 and the First Enzymatic Synthesis of Epoxydammarane from 2,3-Oxidosqualene. Reviewed International journal

    Natsumi Ideno, Shikou Umeyama, Takashi Watanabe, Mami Nakajima, Tsutomu Sato, Tsutomu Hoshino

    Chembiochem : a European journal of chemical biology   19 ( 17 )   1873 - 1886   2018.9

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    The acyclic molecule squalene (1) is cyclized into 6,6,6,6,5-fused pentacyclic hopene (2) and hopanol (3; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene-hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio- and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all-chair conformation. The Ala306 residue in AaSHC is conserved in known squalene-hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5-fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair-chair-chair-boat conformation; in contrast, the normal chair-chair-chair-chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)-2,3-oxidosqualenes (OXSQs) to generate 3α,β-hydroxyhopenes and 3α,β-hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)-OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.

    DOI: 10.1002/cbic.201800281

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  • Bioinspired synthesis of pentacyclic onocerane triterpenoids Reviewed

    Florian Bartels, Young J. Hong, Daijiro Ueda, Manuela Weber, Tsutomu Sato, Dean J. Tantillo, Mathias Christmann

    CHEMICAL SCIENCE   8 ( 12 )   8285 - 8290   2017.12

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    The first chemical synthesis of pentacyclic onocerane triterpenoids has been achieved. A putative biomimetic tricyclization cascade is employed to forge a fused decalin-/oxepane ring system. The synthetic route proceeds to (+)-cupacinoxepin in seven steps and to (+)-onoceranoxide in eight steps in the longest linear sequence, when starting from geranyl chloride and (+)-sclareolide. The bioinspired epoxypolyene cyclization is supported by computational and enzymatic studies.

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  • Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step Reviewed

    Liudmila Tenkovskaia, Mizuki Murakami, Kotone Okuno, Daijiro Ueda, Tsutomu Sato

    CHEMBIOCHEM   18 ( 19 )   1910 - 1913   2017.10

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    Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl--curcumene (C-35) but a bicyclic scaffold from squalene (C-30) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TCY167A produced an unnatural tricyclic triterpenol, but TCY167L, TCY167F, and TCY167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large-scale production of an onoceroid for which natural sources are limited.

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  • Biosynthesis of Sesterterpenes, Head-to-Tail Triterpenes, and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites Reviewed

    Daijiro Ueda, Hiroaki Yamaga, Mizuki Murakami, Yusuke Totsuka, Tetsuro Shinada, Tsutomu Sato

    CHEMBIOCHEM   16 ( 9 )   1371 - 1377   2015.6

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    We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head-to-tail triterpenes, and sesquarterpenes. We have identified an (all-E)-isoprenyl diphosphate synthase (E-IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl--curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E-IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone-7, respectively.

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  • Enzymatic syntheses of unnatural head-to-tail pentacyclic triterpenes by tetraprenyl-beta-curcumene cyclase Reviewed

    Wataru Okamoto, Tsutomu Sato

    TETRAHEDRON LETTERS   54 ( 49 )   6747 - 6750   2013.12

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    Little is known of the biosynthesis of sesquarterpenes and the synthesis of unnatural terpenoids by sesquarterpene biosynthetic enzymes has not yet been reported. In this study, the enzymatic cyclization of head-to-tail acyclic triterpene beta-hexaprene a natural product isolated from Bacillus clausii-using tetraprenyl-beta-curcumene cyclase (TC) from Bacillus subtilis resulted in the formation of two unnatural pentacyclic triterpenes. It was revealed that B. subtilis TC, which forms tetracyclic terpenoid scaffold from tetraprenyl-beta-curcumene in vivo, could be used to construct the 6/6/6/6/6-fused pentacyclic scaffold in vitro, suggesting that the active site cavity of TC has sufficient space to accommodate this unnatural pentacyclic scaffold. This is the first report demonstrating the utility of a sesquarterpene cyclase toward the synthesis of unnatural terpenoids. (C) 2013 Elsevier Ltd. All rights reserved.

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  • Identification of Novel Sesterterpene/Triterpene Synthase from Bacillus clausii Reviewed

    Tsutomu Sato, Hiroaki Yamaga, Shoji Kashima, Yusuke Murata, Tetsuro Shinada, Chiaki Nakano, Tsutomu Hoshino

    CHEMBIOCHEM   14 ( 7 )   822 - 825   2013.5

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    DOI: 10.1002/cbic.201300035

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  • Insight into the pathway for biosynthesis of sesquarterpenes in nonpathogenic Mycobacterium species: identification of heptaprenylcycli-14E,18E-diene Reviewed

    Tsutomu Sato, Eri Ono, Mami Nakajima, Chiaki Nakano, Tsutomu Hoshino

    TETRAHEDRON LETTERS   53 ( 20 )   2522 - 2524   2012.5

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    A new sesquarterpene, heptaprenylcycli-14E,18E-diene, was isolated from Mycobacterium chlorophenolicum cells grown up to the stationary phase. The absence of heptaprenylcycli-14Z,18E-diene indicates that only (E,E,E)-geranylgeranyl diphosphate may be utilized as an intermediate of sesquarterpene biosynthesis in the stationary phase, in contrast with the logarithmic growth phase in which both (E,E)-farnesyl diphosphate and (E,E,E)-geranylgeranyl diphosphate are used. Further, our findings suggest that the step-wise reduction of the polyprenyl group in sesquarterpene biosynthesis might proceed in a different order from that in chlorophyll biosynthesis. (C) 2012 Elsevier Ltd. All rights reserved.

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  • Triterpene Cyclases from Oryza sativa L.: Cycloartenol, Parkeol and Achilleol B Synthases Reviewed

    Ryousuke Ito, Kouya Mori, Ippei Hashimoto, Chiaki Nakano, Tsutomu Sato, Tsutomu Hoshino

    ORGANIC LETTERS   13 ( 10 )   2678 - 2681   2011.5

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    The gene products of AK121211, AK066327, and AK070534 from Oryza sativa encode cycloartenol, parkeol, and achilleol B synthases, respectively. Parkeol synthase is a unique enzyme that affords parkeol as a single product. Achilleol B synthase is the third seco-type triterpene cyclase identified to date, and triterpenes produced by this synthase include achilleol B (90%), tetracyclic (5.12%) and pentacyclic scaffolds (4.37%), and unidentified triterpenes (0.51%). The pathway for achilleol B biosynthesis is proposed.

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  • Characterization of the Ry3378c Gene Product, a New Diterpene Synthase for Producing Tuberculosinol and (13R, S)-Isotuberculosinol (Nosyberkol), from the Mycobacterium tuberculosis H37Rv Genome Reviewed

    Chiaki Nakano, Takahiro Ootsuka, Kazutoshi Takayama, Toshiaki Mitsui, Tsutomu Sato, Tsutomu Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 1 )   75 - 81   2011.1

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    The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of H-1- and C-13-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.

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  • Insight into C-35 terpene Biosyntheses by Nonpathogenic Mycobacterium Species: Functional Analyses of Three Z-Prenyltransferases and Identification of Dehydroheptaprenylcyclines Reviewed

    Tsutomu Sato, Kazuo Takizawa, Yuriko Onto, Hanayo Kudo, Tsutomu Hoshino

    CHEMBIOCHEM   11 ( 13 )   1874 - 1881   2010.9

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    Nonpathogenic Mycobacterium species produce rare cyclic C-35 terpenes that are biosynthesized by cyclization of Z-type C-35 polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C-35 terpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z-prenyltransferase, biosynthesizes C-35-heptaprenyl diphosphate as a main product from (E,E)-farnesyl diphosphate (E,E-FPP) and (E,E,E)-geranylgeranyl diphosphate (E,E,E-GGPP), but produces a C-50-decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C-35 terpenes, (14E)- and (14Z)-dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C-35 terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of C-35 terpenes could reduce both E and Z prenyl residues.

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  • Substrate specificity of the CYC2 enzyme from Kitasatospora griseola: production of sclarene, biformene and novel bicyclic diterpenes by the enzymatic reactions of labdane- and halimane-type diterpene diphosphates Reviewed

    Chiaki Nakano, Tsutomu Hoshino, Tsutomu Sato, Tomonobu Toyomasu, Tohru Dairi, Takeshi Sassa

    TETRAHEDRON LETTERS   51 ( 1 )   125 - 128   2010.1

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    The enzymatic reaction of copalyl diphosphate (CDP), ent-CDP, syn-CDP, and tuberculosinyl diphosphate with the CYC2 enzyme from Kitasatospora griseola afforded sclarene, biformene, and novel diterpenes having a buta-1,3-diene moiety in the side chain. The substrate specificity of the CYC2 is discussed. (C) 2009 Elsevier Ltd. All rights reserved.

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  • Novel Compounds of Octahydroheptaprenyl Mycolic Acyl Ester and Monocyclic C-35-Terpene, Heptaprenylcycline B, from Non-Pathogenic Mycobacterium Species Reviewed

    Tsutomu Sato, Ryosuke Takagi, Yuriko Orito, Eri Ono, Tsutomu Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   74 ( 1 )   147 - 151   2010.1

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    A search for C-35-terpenes from non-saponified extracts of 12 non-pathogenic Mycobacterium species was carried out. Octahydroheptaprenyl mycolic acyl esters were isolated from M. chlorophenolicum cells which were also found from M. thermoresistibile, M. vanbaalenii, M. aichiense, M. smegmatis, and M. parafortuitum. This is the first report on a polyprenol esterified by a mycolate. A novel monocyclic C-35-terpene possessing a ketone, named heptaprenylcycline B, was isolated, which was detected from M. chlorophenolicum and M. vanbaalenii. The biosynthetic pathway to heptaprenylcycline B was investigated with ancymidol which acts as an inhibitor of a P450 monooxygenase. This experiment suggested that the P450 monooxygenase may be responsible for the production of heptaprenylcycline B.

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  • Reviewing the Polyolefin Cyclization Reaction of the C-35 Polyprene Catalyzed by Squalene-Hopene Cyclase Reviewed

    Tsutomu Hoshino, Yuko Kumai, Tsutomu Sato

    CHEMISTRY-A EUROPEAN JOURNAL   15 ( 9 )   2091 - 2100   2009

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    Squalene-hopene cyclase (SHC) catalyzes the polycyclization of squalene (C-30) to the pentacyclic hopene With regio- and stereochemical specificity. In this study, we reviewed the polycyclization reaction of the C-35 polyprenoid catalyzed by SHC. Surprisingly, our results completely disagreed with a previous publication in which it was reported that a hexacyclic skeleton was constructed as the single product in 10% yield (I. Abe. H. Tanaka, H. Noguchi, J. Am. Chem. Soc. 2002, 124, 14514-14515). Our experimental results showed that many tri- and tetracyclic products, up to 12. including novel carbocyclic cores, were generated in a high conversion ratio (97%), but no detectable amounts of the penta- and hexacycle were produced. The mechanisms for the formation of the C-31 Polyprene products isolated by us are discussed in this paper. The following four conformations were generated during the polycyclization cascade: chair-chair-boat, chair-chair-chair, chairchair-chair-boat, and chair-chair-chair-chair. Larger amounts of the false intermediates with 13 alpha-H (tricycle) and 17 alpha-H (tetracycle) were produced compared with the true intermediates (13 beta-H and 17 beta-H), which indicates that the C-35 polyprene cannot fold correctly in the enzyme cavity due to the extra C-5 unit appended to squalene. This Would have promoted the formation of the aborted cyclization products With tri- and tetracycles. In addition, the fact that no penta- or hexacyclic products were formed further indicates that SHC does not have sufficient space to accommodate the entire carbon framework of C-31.

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  • Biosynthesis of a novel cyclic C-35-terpene via the cyclisation of a Z-type C-35-polyprenyl diphosphate obtained from a nonpathogenic Mycobacterium species Reviewed

    Tsutomu Sato, Atsushi Kigawa, Ryosuke Takagi, Tomomi Adachi, Tsutomu Hoshino

    ORGANIC & BIOMOLECULAR CHEMISTRY   6 ( 20 )   3788 - 3794   2008.10

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    Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C35-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of I was considerably greater at a higher temperature of 37 degrees C than at an optimal temperature of 28 degrees C, while that of 2 remained unchanged at all temperatures. A feeding experiment with D-[1(-13) C]glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C-35-isoprenoid. The substrate 2-PP for C-35-terpene cyclase has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds as the natural substrate has not yet been detected. Despite a careful search using the FASTA3 program, we could not detect any gene that is homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

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  • Sterol biosynthesis by a prokaryote: First in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus Reviewed

    Chiaki Nakano, Akihiro Motegi, Tsutomu Sato, Masayuki Onodera, Tsutomu Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 10 )   2543 - 2550   2007.10

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    Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.

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  • Biosynthesis of violacein: a genuine intermediate, protoviolaceinic acid, produced by VioABDE, and insight into VioC function Reviewed

    Kouhei Shinoda, Takuji Hasegawa, Hiroaki Sato, Masaaki Shinozaki, Hirotomo Kuramoto, Yosuke Takamiya, Tsutomu Sato, Naoki Nikaidou, Takeshi Watanabe, Tsutomu Hoshino

    CHEMICAL COMMUNICATIONS   ( 40 )   4140 - 4142   2007

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    A biosynthetic intermediate of violacein produced by the mixed enzymes of VioABDE was elucidated to be 5-( 5- hydroxy1H- indol- 3- yl)- 3-( 1H- indol- 3- yl)- 1H- pyrrole- 2- carboxylic acid, named protoviolaceinic acid, indicating that VioC, responsible for the final biosynthetic step, works to oxygenate at the 2- position of the right side indole ring, and that the oxygenation reaction to form the central pyrrolidone core proceeds in a nonenzymatic fashion.

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  • P-295 NOVEL DITERPENE SYNTHASES FROM MYCOBACTERIUM TUBERCULOSIS H37RV

    Nakano Chiaki, Sato Tsutomu, Hoshino Tsutomu

    International Symposium on the Chemistry of Natural Products   2006   "P - 295"   2006.7

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    DOI: 10.24496/intnaturalprod.2006.0__P-295_

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  • Mycobacterium tuberculosis H37Rv3377c encodes the diterpene cyclase for producing the halimane skeleton Reviewed

    C Nakano, T Okamura, T Sato, T Dairi, T Hoshino

    CHEMICAL COMMUNICATIONS   ( 8 )   1016 - 1018   2005.2

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    The cloning and functional expression of Mycobacterium tuberculosis Rv3377c in Escherichia coli revealed that this gene encodes the diterpene cyclase for producing (+)-5(6),13-halimadiene-15-ol, which accepts geranylgeranyldiphosphate as the intrinsic substrate.

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  • Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius Reviewed

    T Sato, M Kouda, T Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   68 ( 3 )   728 - 738   2004.3

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    To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules ("front water" and "back waters") were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two, residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the "front water molecule," that is, to place the "front water" in a favorable position, leading to the minimal production of by-products, i.e., hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the "back waters" is constructed, increase the polarization of the "front water" to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.

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  • Deletion of the Gly600 residue of alicyclobacillus acidocaldarius squalene cyclase alters the substrate specificity into that of the eukaryotic-type cyclase specific to (3S)-2,3-oxidosqualene Reviewed

    T Hoshino, K Shimizu, T Sato

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   43 ( 48 )   6700 - 6703   2004

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  • Squalene-hopene cyclase: final deprotonation reaction, conformational analysis for the cyclization of (3R,S)-2,3-oxidosqualene and further evidence for the requirement of an isopropylidene moiety both for initiation of the polycyclization cascade and for the formation of the 5-membered E-ring Reviewed

    T Hoshino, S Nakano, T Kondo, T Sato, A Miyoshi

    ORGANIC & BIOMOLECULAR CHEMISTRY   2 ( 10 )   1456 - 1470   2004

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    To provide insight into the polycyclization mechanism of squalene by squalene-hopene cyclase (SHC) from Alicyclobacilus acidocaldarius, some analogs of nor- and bisnorsqualenes were synthesized including the deuterium-labeled squalenes and incubated with the wild-type SHC, leading to the following inferences. (1) The deprotonation reaction for the introduction of the double bond of the hopene skeleton occurs exclusively from the Z-methyl group on the terminal double bond of squalene. (2) 3R-Oxidosqualene was folded in a boat conformation for the A-ring construction, while the 3S-form was in a chair structure. (3) The terminal two methyl groups are indispensable both for the formation of the 5-membered E-ring of the hopene skeleton and for the initiation of the polycyclization cascade, but the terminal Z-methyl group has a more crucial role for the construction of the 5-membered E-ring than the E-methyl group. (4) Some of the novel terpene skeletons, 36, 37, 39 and 40, were created from the analogs employed in this investigation.

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  • Functional analyses of Tyr420 and Leu607 of Alicyclobacillus acidocaldarius squalene-hopene cyclase. Neoachillapentaene, a novel triterpene with the 1,5,6-trimethylcyclohexene moiety produced through folding of the constrained boat structure Reviewed

    T Sato, S Sasahara, T Yamakami, T Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   66 ( 8 )   1660 - 1670   2002.8

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    The functions of Tyr420 and Leu607 were analyzed by constructing various site-directed mutants. The mutation at position 420 into Ala and Gly gave bicyclic alpha-and gamma-polypodatetraene in significant amounts, but with a trace amount of tricyclic malabaricatriene. The kinetic data for and the product distribution of the Y420F mutant indicate that the major function of Tyr420 is to stabilize the 6/6-fused bicyclic cation. Mutation experiments on Leu607 demonstrate that the appropriate steric bulk size at position 607 is required to strongly bind with the product-like conformation formed during the polycyclization process. Introduction of the bulkiest Trp residue into 420 or 607 led to the production of a novel monocyclic triterpene having the (5R,6R)-1,5,6-trimethylcyclohexene ring, named neoachillapentaene, indicating that the enzymatic cyclization proceeded via a constrained boat structure. Folding of the squalene molecule into a boat conformation by squalene cyclase has not been reported before.

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  • Catalytic function of the residues of phenylalanine and tyrosine conserved in squalene-hopene cyclases Reviewed

    T Sato, T Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   65 ( 10 )   2233 - 2242   2001.10

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    Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-pi interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three residues, Y372, V474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.

    DOI: 10.1271/bbb.65.2233

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  • Functional analysis of Phe605, a conserved aromatic amino acid in squalene-hopene cyclases Reviewed

    T Hoshino, M Kouda, T Abe, T Sato

    CHEMICAL COMMUNICATIONS   ( 16 )   1485 - 1486   2000

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    Incubation of squalene with the site-directed mutant F605A of squalene-hopene cyclase from Alicyclobacillus acidocaldarius yielded many triterpenes consisting of the 6/6/5-fused tri-, 6/6/6/5-fused tetra-, and 6/6/6/6/5-fused pentacyclic skeletons, the function of F605 being assignable for facilitating the ring expansion and for stabilizing the hopanyl C22-cation, possibly via cation-pi interactions.

    DOI: 10.1039/b004129g

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  • Functional analysis of the DXDDTA motif in squalene-hopene cyclase by site-directed mutagenesis experiments: Initiation site of the polycyclization reaction and stabilization site of the carbocation intermediate of the initially cyclized A-ring Reviewed

    T Sato, T Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   63 ( 12 )   2189 - 2198   1999.12

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    In order to clarify the function of the DXDDTA motif in squalene-hopene cyclase and to identify the acidic amino acid residues crucial for the catalysis, site-directed mutagenesis experiments were carried out. The following results were found: (1) residues D374 and D376 work for the initiation of polyolefin cyclization which arises from the proton attack on the terminal double bond; (2) residue D377 stabilizes C-10 carbocation of the initially cyclized A-ring intermediate, leading to subsequent B-ring closure, which was further verified by isolating the partially cyclized monocyclic product; (3) residues D313 and D447 outside the DXDDTA motif were identified as new active sites; (4)the H451 residue is likely to work in the protonated form to enhance the acidity of the carboxyl groups of D374 and/or D376.

    DOI: 10.1271/bbb.63.2189

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  • Functional analysis of phenylalanine 365 in hopene synthase, a conserved amino acid in the families of squalene and oxidosqualene cyclases Reviewed

    T Hoshino, T Sato

    CHEMICAL COMMUNICATIONS   ( 19 )   2005 - 2006   1999.10

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    Two bicyclic products were accumulated by the mutant F365A, showing the amino acid residue is located close to the transient C-8 carbocation intermediate in the active site cavity; the mutants of F365Y and F365W significantly accelerated the cyclization reaction at low temperatures.

    DOI: 10.1039/a905559b

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  • Kinetic studies on the function of all the conserved tryptophans involved inside and outside the QW motifs of squalene-hopene cyclase: Stabilizing effect of the protein structure against thermal denaturation Reviewed

    T Sato, T Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   63 ( 7 )   1171 - 1180   1999.7

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    Site-directed mutagenesis experiments were carried out to identify the responsibility of the eight QW motifs for the reaction catalyzed by squalene-hopene cyclase (SHC). Alterations of the conserved tryptophans, which are responsible for the stacking structure with glutamine, into aliphatic amino acids gave a significantly lower temperature for the catalytic optimum as for the mutageneses of QW motifs 4, 5a and 5b, which are specifically present in SHCs. However, there was no change in the optimal temperatures of the mutated SHCs targeted at the other five motifs 1, 2, 3, 5c and 6. Thus, reinforcement against heat denaturation can be proposed as a function of the three QW motifs 4, 5a and 5b, but no function could be identified for the QW motifs 1, 2, 3, 5c and 6, although they are commonly found in all the families of prokaryotic SHCs and eukaryotic oxidosqualene cyclases. On the other hand, the three conserved tryptophans of W169, W312 and W489, which are located inside the putative central cavity and outside the QW motifs, were identified as components of the active sites, but also had a function against thermal denaturation. The other two tryptophan residues of W142 and W558, which are located outside the QW motifs, were found not to be active sites, but also had a role for stabilizing the protein structure. It is noteworthy that the mutants replaced by phenylalanine had higher temperatures for the catalytic optimum than those replaced by aliphatic amino acids. The catalytic optimal pH values for all the mutants remained unchanged with an identical value of 6.0.

    DOI: 10.1271/bbb.63.1171

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  • On the cyclization mechanism of squalene: a ring expansion process of the five-membered D-ring intermediate Reviewed

    T Sato, T Abe, T Hoshino

    CHEMICAL COMMUNICATIONS   ( 23 )   2617 - 2618   1998.12

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    Site-directed mutagenesis experiments with W169F, W169H and W489F for the squalene-hopene cyclase, and the formation of 10 possessing the five-membered D-ring and a tetrahydrofuran moiety as the enzyme product of the analogue 8 with a hydroxy group, strongly suggest that a ring expansion reaction from the five- to the six-membered ring is responsible for the D-ring formation of hopene.

    DOI: 10.1039/a806948d

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  • Overexpression of squalene-hopene cyclase by the pET vector in Escherichia coli and first identification of tryptophan and aspartic acid residues inside the QW motif as active sites Reviewed

    T Sato, Y Kanai, T Hoshino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   62 ( 2 )   407 - 411   1998.2

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    An overexpression system for squalene-hopene cyclase (SHC) was constructed by using the pET3a vector, which is responsible for high expression with help from the strong T7 promoter when incorporated into E. coli BL21(DE3). Site-directed mutagenesis experiments prove that two amino acid residues of tryptophan and aspartic acid inside the QW-motif 5 resided as active sites.

    DOI: 10.1271/bbb.62.407

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Books

  • 海棲哺乳類大全 : 彼らの体と生き方に迫る

    田島, 木綿子, 山田, 格

    緑書房  2021.3  ( ISBN:9784895315883

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MISC

  • 抗酸菌の新しいタイプのZ、E混成型プレニル基還元酵素の同定と機能解析(Identification and functional analysis of a new type of Z,E-mixed prenyl reductase from mycobacteria)

    阿部 透, 袴田 真理子, 西山 晃史, 立石 善隆, 松本 壮吉, 邊見 久, 上田 大次郎, 佐藤 努

    日本細菌学雑誌   77 ( 1 )   68 - 68   2022.2

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  • 龍涎香の人工合成系の確立に向けてー新規アンブレイン合成酵素の創出ー Reviewed

    佐藤努

    化学と生物   59 ( 5 )   222 - 224   2021.5

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  • Development of ambrein synthase and investigation of some biological activities of ambrein.

    上田大次郎, 佐藤努

    Aroma Research   22 ( 2 )   2021

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  • 龍涎香の主成分アンブレインの人工生合成経路の創出

    上田大次郎, 佐藤努

    バイオサイエンスとインダストリー   79 ( 3 )   2021

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  • マイコバクテリア由来新規Z,E混成型プレニル基還元酵素の機能解析によるセスクアテルペン生合成の洞察

    阿部透, 袴田真理子, 西山晃史, 立石善隆, 松本壮吉, 邊見久, 上田大次郎, 佐藤努

    イソプレノイド研究会例会講演要旨集   31st (CD-ROM)   2021

  • トリテルペン/セスクアテルペン環化酵素の立体構造解析

    亀谷太一, 久保田智巳, 品田哲郎, 上田大次郎, 佐藤努

    イソプレノイド研究会例会講演要旨集   31st (CD-ROM)   2021

  • Conversion of enzyme-synthesized ambrein to aroma components and pharmacological activity of ambrein

    川越幸奈, 上田大次郎, 柿原嘉人, 原崇, 佐藤努

    日本農芸化学会大会講演要旨集(Web)   2021   2021

  • Functional analysis of E-isoprenyl diphosphate synthases from Mycobacterium tuberculosis

    阿部透, 上田大次郎, 佐藤努

    日本農芸化学会大会講演要旨集(Web)   2021   2021

  • 抗酸菌由来E型イソプレニル二リン酸合成酵素群の機能解析

    阿部透, 上田大次郎, 佐藤努

    日本放線菌学会大会講演要旨集   35th (CD-ROM)   2021

  • Analysis of cyclic structure formation mechanism of class IB terpene synthase

    浅田和也, 上田大次郎, 三木邦夫, 藤橋雅宏, 品田哲郎, 佐藤努

    日本農芸化学会大会講演要旨集(Web)   2021   2021

  • マイコバクテリア由来新規Z,E混成型プレニル基還元酵素の機能解析

    阿部透, 袴田真理子, 西山晃史, 立石善隆, 松本壮吉, 邊見久, 上田大次郎, 佐藤努

    日本放線菌学会大会講演要旨集   35th (CD-ROM)   2021

  • 抗酸菌由来E型イソプレニル二リン酸合成酵素の機能解析によるイソプレノイド生合成の洞察

    阿部透, 上田大次郎, 佐藤努

    日本農芸化学会関東支部講演要旨集(CD-ROM)   2020   2020

  • クラスIB大型テルペン合成酵素の基質複合体の結晶構造

    金本壮平, 佐藤努, 品田哲朗, 三木邦夫, 深井周也, 藤橋雅宏

    日本結晶学会年会講演要旨集   2020 (CD-ROM)   2020

  • 人工龍涎香合成系の構築とアンブレインの新規生物活性の解析

    山辺陽太, 川越幸奈, 上田大次郎, 柿原嘉人, 原崇, 佐藤努

    イソプレノイド研究会例会講演要旨集   30th   2020

  • ビタミンK生合成機構における「酵素」と「非酵素」による側鎖切断メカニズムの解明

    廣田 佳久, 須原 義智, 佐藤 努, 上田 大次郎

    特別教育・研究報告集   279 - 282   2019

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  • クラスIBテルペン合成酵素の触媒反応の解析

    西智之, STEPANOVA Rafaella, 菅原啓, 上田大次郎, 藤橋雅宏, 三木邦夫, 保野陽子, 品田哲郎, 佐藤努

    日本農芸化学会大会講演要旨集(Web)   2019   2019

  • Class IB大型テルペン合成酵素の基質認識機構の解析

    藤橋雅宏, 稲木隼人, 西智之, 上田大次郎, 佐藤努, 品田哲朗, 三木邦夫

    イソプレノイド研究会例会講演要旨集   29th   2019

  • アンブレインの香気成分への変換および破骨細胞分化誘導活性

    川越幸奈, 上田大次郎, 柿原嘉人, 佐藤努

    イソプレノイド研究会例会講演要旨集   29th   2019

  • Mycobacterium属細菌由来新規ヘプタプレニル還元酵素の同定とZ型セスクアテルペン環化酵素の探索

    阿部透, 尾崎真夢, 吉田優里, 三浦彩奈, 相良昌寛, 上田大次郎, 上田大次郎, 金古堅太郎, 三ツ井敏明, 三ツ井敏明, 佐藤努, 佐藤努

    日本放線菌学会大会講演要旨集   34th   2019

  • Analysis of ligand-recognition mechanism of class IB terpene synthase

    稲木隼人, 佐藤努, 保野陽子, 品田哲郎, 三木邦夫, 藤橋雅宏

    量子ビームサイエンスフェスタ(Web)   2018   2019

  • Novel and unnatural terpenes found from sesquarterpene cyclase study

    Ueda Daijiro, Yamaga Hiroaki, Okamoto Wataru, Totsuka Yusuke, Shinada Tetsuro, Hoshino Tsutomu, Sato Tsutomu

    Symposium on the Chemistry of Natural Products, symposium papers   56 ( 0 )   2018.7

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    &lt;p&gt;Biosynthetic studies on the sesquarterpenes, a family of C&lt;sub&gt;35 &lt;/sub&gt;terpenes, in Bacillus subtilis have identified a new type of terpene cyclase, tetraprenyl-b-curcumene synthase (TS), which lacks sequence homology to any known terpene synthases. We recently revealed that Bacillus megaterium tetraprenyl-b-curcumene cyclase (TC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail C&lt;sub&gt;35&lt;/sub&gt; 1 and tail-to-tail C&lt;sub&gt;30&lt;/sub&gt; 3 into pentacyclic 2 and bicyclic 4, respectively, in vivo (Scheme 1). In this presentation, we report novel and unnatural terpenes found from sesquarterpene cyclase (TS and TC) study.&lt;/p&gt;&lt;p&gt;1) We discovered head-to-tail types of novel acyclic sesterterpene 5 and triterpene 6 in B. clausii, and revealed that compounds 5 and 6 are biosynthesized by TS homolog (Bcl-TS) from GFPP and HexPP, respectively (Scheme 2). The present study revealed that TS homologs are a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. Genome mining of this new terpene synthase family will lead to discoveries of novel terpene synthases and terpenoids in the future.&lt;/p&gt;&lt;p&gt;2) The enzymatic cyclization of head-to-tail acyclic triterpene 6isolated from Bacillus clausii using B. subtilis TC resulted in the formation of two unnatural pentacyclic triterpenes 7and 8 (Scheme 3). It was revealed that B. subtilis TC, which forms tetracyclic terpenoid scaffold from tetraprenyl-β-curcumene in vivo, could be used to construct the 6/6/6/6/6-fused pentacyclic scaffold in vitro, suggesting that the active site cavity of TC has sufficient space to accommodate this unnatural pentacyclic scaffold. &lt;/p&gt;&lt;p&gt;3) We revealed that TC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase (Scheme 4). Also, we report the first onoceroids from bacterial origin. Our discoveries suggested that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of TC enabled the synthesis of (+)-ambrein 11, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase–catalyzed reaction from easily available squalene (Scheme 5).&lt;/p&gt;

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  • Large‐terpene合成酵素の酵素的諸性質の解析と部位特異的変異

    西智之, 菅原啓, 小川佳央, 高橋宏忠, 上田大次郎, 藤橋雅宏, 三木邦夫, 保野陽子, 品田哲郎, 佐藤努

    日本蛋白質科学会年会プログラム・要旨集   18th   50   2018.5

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  • Large‐terpene合成酵素の酵素的諸性質の解析と部位特異的変異

    菅原啓, 西智之, 小川佳央, 高橋宏忠, 上田大次郎, 藤橋雅宏, 三木邦夫, 保野陽子, 品田哲郎, 佐藤努

    日本農芸化学会大会講演要旨集(Web)   2018   ROMBUNNO.3A17p02 (WEB ONLY)   2018.3

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  • Large‐terpene合成酵素の結晶構造解析

    藤橋雅宏, 佐藤努, 田中勇真, 山本大輔, 西智之, 上田大次郎, 村上瑞気, 保野陽子, 関原あい, 福和真, 品田哲郎, 三木邦夫

    日本農芸化学会大会講演要旨集(Web)   2018   ROMBUNNO.3A17p01 (WEB ONLY)   2018.3

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  • 新規ファミリーに属するテルペン合成酵素の基質認識部位の同定

    稲木隼人, 佐藤努, 保野陽子, 品田哲郎, 三木邦夫, 藤橋雅宏

    日本結晶学会年会講演要旨集   2018   2018

  • Large-terpene合成酵素の構造と機能

    藤橋雅宏, 佐藤努, 田中勇真, 山本大輔, 西智之, 上田大次郎, 村上瑞気, 保野陽子, 関原あい, 福和真, 品田哲朗, 三木邦夫

    日本蛋白質科学会年会プログラム・要旨集   18th   2018

  • Large-terpene合成酵素の酵素学的諸性質の解析と部位特異的変異

    西智之, 菅原啓, 小川佳央, 高橋宏忠, 上田大次郎, 藤橋雅宏, 三木邦夫, 保野陽子, 品田哲郎, 佐藤努

    香料・テルペンおよび精油化学に関する討論会講演要旨集   62nd   2018

  • Bacillus属細菌由来テルペノイドのユニークな生合成経路の解析

    上田大次郎, 松ヶ根沙織, 西智之, 菅原啓, 岡本渉, 藤橋雅宏, 三木邦夫, 橋本昌征, 保野陽子, 品田哲郎, 佐藤努

    イソプレノイド研究会例会講演要旨集   28th   2018

  • Large-terpene合成酵素の構造機能相関

    藤橋雅宏, 佐藤努, 田中勇真, 山本大輔, 西智之, 上田大次郎, 村上瑞気, 保野陽子, 関原あい, 福和真, 品田哲朗, 三木邦夫

    イソプレノイド研究会例会講演要旨集   28th   2018

  • Study on Ambergris

    上田大次郎, 佐藤努

    季刊香料   ( 273 )   53‐59   2017.3

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  • Study on Ambergris

    上田 大次郎, 佐藤 努

    香料   ( 273 )   53 - 59   2017

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  • 龍涎香の主成分アンブレインの酵素合成

    上田大次郎, 星野力, 佐藤努

    Aroma Res   16 ( 2 )   142 - 143   2015.5

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  • 3P-269 Biosynthesis of rare terpenoids in Bacillus clausii

    Ueda Daijiro, Yamaga Hiroaki, Murakami Mizuki, Totsuka Yusuke, Shinada Tetsuro, Sato Tsutomu

    日本生物工学会大会講演要旨集   67   338 - 338   2015

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    Other Link: http://dl.ndl.go.jp/info:ndljp/pid/10536373

  • 龍涎香の主成分アンブレインの酵素合成

    上田大次郎, 星野力, 佐藤努

    バイオサイエンスとインダストリー   72 ( 5 )   410 - 411   2014.9

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  • Novel and unnatural terpenes found from sesquarterpene cyclase study

    Ueda Daijiro, Yamaga Hiroaki, Okamoto Wataru, Totsuka Yusuke, Shinada Tetsuro, Hoshino Tsutomu, Sato Tsutomu

    Symposium on the Chemistry of Natural Products, symposium papers   56 ( 0 )   Oral18   2014

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    <p>Biosynthetic studies on the sesquarterpenes, a family of C<sub>35 </sub>terpenes, in Bacillus subtilis have identified a new type of terpene cyclase, tetraprenyl-b-curcumene synthase (TS), which lacks sequence homology to any known terpene synthases. We recently revealed that Bacillus megaterium tetraprenyl-b-curcumene cyclase (TC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail C<sub>35</sub> 1 and tail-to-tail C<sub>30</sub> 3 into pentacyclic 2 and bicyclic 4, respectively, in vivo (Scheme 1). In this presentation, we report novel and unnatural terpenes found from sesquarterpene cyclase (TS and TC) study.</p><p>1) We discovered head-to-tail types of novel acyclic sesterterpene 5 and triterpene 6 in B. clausii, and revealed that compounds 5 and 6 are biosynthesized by TS homolog (Bcl-TS) from GFPP and HexPP, respectively (Scheme 2). The present study revealed that TS homologs are a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. Genome mining of this new terpene synthase family will lead to discoveries of novel terpene synthases and terpenoids in the future.</p><p>2) The enzymatic cyclization of head-to-tail acyclic triterpene 6isolated from Bacillus clausii using B. subtilis TC resulted in the formation of two unnatural pentacyclic triterpenes 7and 8 (Scheme 3). It was revealed that B. subtilis TC, which forms tetracyclic terpenoid scaffold from tetraprenyl-β-curcumene in vivo, could be used to construct the 6/6/6/6/6-fused pentacyclic scaffold in vitro, suggesting that the active site cavity of TC has sufficient space to accommodate this unnatural pentacyclic scaffold. </p><p>3) We revealed that TC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase (Scheme 4). Also, we report the first onoceroids from bacterial origin. Our discoveries suggested that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of TC enabled the synthesis of (+)-ambrein 11, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase–catalyzed reaction from easily available squalene (Scheme 5).</p>

    DOI: 10.24496/tennenyuki.56.0_Oral18

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  • セスクアテルペン環化酵素の部位特異的変異および非天然型テルペンの創出

    上田大次郎, 岡本渉, 山本彩乃, 遠塚悠輔, 品田哲郎, 仲野千秋, 佐藤努

    香料・テルペンおよび精油化学に関する討論会講演要旨集   57th   369 - 371   2013.10

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  • Unique biosynthesis of sesquarterpenes (C35 terpenes) Reviewed

    Tsutomu Sato

    Bioscience, Biotechnology and Biochemistry   77 ( 6 )   1155 - 1159   2013

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    To the best of my knowledge, only 19 cyclic and 8 linear C35 terpenes have been identified to date, and no family name was assigned to this terpene class until recently. In 2011, it was proposed that these C35 terpenes should be called sesquarterpenes. This review highlights the biosynthesis of two kinds of sesquarterpenes (C35 terpenes) that are produced via cyclization of a linear C35 isoprenoid in Bacillus and Mycobacterium species. In Bacillus species, a new type of terpene cyclase that has no sequence homology with any known terpene synthases, as well as a bifunctional terpene cyclase that biosynthesizes two classes of cyclic terpenes with different numbers of carbons as natural products, have been identified. On the other hand, in Mycobacterium species, the first bifunctional Z-prenyltransferase has been found, but a novel terpene cyclase and a unique polyprenyl reductase remain unidentified. The identification of novel enzyme types should lead to the discovery of many homologous enzymes and their products including novel natural compounds. On the other hand, many enzymes responsible for the biosynthesis of natural products have low substrate specificities in vitro. Therefore, to find novel natural products present in organisms, the multifunctionality of enzymes in the biosynthetic pathway of natural products should be analyzed.

    DOI: 10.1271/bbb.130180

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  • 植物毒素チコリン生合成遺伝子の全塩基配列の決定

    佐藤努, 佐藤努, 山之内健太, 若林広恵, 橋本昌征, 兼目裕充, 浅川義範, 白川隆, 仲野千秋, 星野力, 星野力

    日本農芸化学会大会講演要旨集(Web)   2013   2013

  • ユニークなセスクアテルペン(C_<35>テルペン)生合成経路 : 今までに類のない新型と二機能性テルペン環化酵素の同定

    佐藤 努

    化学と生物   50 ( 9 )   622 - 623   2012.9

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    DOI: 10.1271/kagakutoseibutsu.50.622

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  • メタゲノム由来モジュールライブラリーを用いた非天然型ペプチド創出

    佐藤努, 山之内健太, 若林広恵, 橋本昌征, 内山拓, 兼目裕充, 浅川義範, 白川隆, 仲野千秋, 星野力

    日本農芸化学会関東支部講演要旨集   2012 ( Oct )   2012

  • 17 Sesquarterpenes (C_<35> Terpenes) from Bacteria : Identifications of Unique Biosynthetic Enzymes and Search for Novel Natural Products(Oral Presentation)

    Sato Tsutomu, Nakajima Mami, Hoshino Tsutomu, Hoshino Hiroko, Yoshida Satoru, Takizawa Kazuo, Orito Yuriko, Takagi Ryosuke, Tanno Mizuki, Kudo Hanayo, Ono Eri

    Symposium on the Chemistry of Natural Products, symposium papers   53 ( 0 )   97 - 102   2011

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    In this study, mono- and pentacyclic C_<35> terpenes from Bacillus subtilis were biosynthesized via the cyclization of C_<35> isoprenoid by using purified enzymes, including the first identified new terpene cyclase, tetrapreny1-13-curcumene synthase, that shows no sequence homology to any of the known terpene cyclases. Based on these findings, we propose that these C_<35> terpenes are called the new family of "sesquarterpenes." This study demonstrated that a tetrapreny1-β-curcumene cyclase (TC) from B. subtilis, which was originally identified as a sesquarterpene cyclase converting a head-to-tail type of monocycle to a pentacycle, also cyclized a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-Hydroxypolypoda-13,17,21-triene. The 8α-Hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium, suggesting that the TC is bifunctional, cyclizing both tetrapreny1-α-curcumene and squalene in vivo. This is the first report describing the bifunctional terpene cyclase, which biosynthesizes 2 classes of cyclic terpenes with different numbers of carbons as natural products in the organism. Non-pathogenic Mycobacterium species also produce cyclic sesquarterpenes, which are biosynthesized via cyclization of Z-type C_<35> polyprenyl diphosphate. To provide deeper insight into the biosynthesis of sesquarterpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M vanbaalenii identified by genomic analysis. Mvan_3822, a novel bi-functional Z-prenyltransferase, biosynthesizes C_<35>-heptaprenyl diphosphate as a main product from E,E-FPP and E,E,E-GGPP, but produces a C_<50>-decaprenyl diphosphate from GPP. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C_<35>-terpenes, 14E- and 14Z-dehydroheptaprenylcycline, were identified as minor metabolites in non-pathogenic Mycobacterium cells. Sesquarterpenes could be biosynthesized by two routes, in which E- and Z-geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of sesquarterpenes may work to reduce both E- and Z-prenyl residues. The studies on sesquarterpenes promise to be an attractive field for expanding our understanding of the terpene world.

    DOI: 10.24496/tennenyuki.53.0_97

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  • P-85 Biosynthesis of a novel cyclic C_<35>-terpene by the cyclisation of a Z-type C_<35>-polyprenyl diphosphate obtained from a nonpathogenic Mycobacterium species(Poster Presentation)

    Sato Tsutomu, Kigawa Atsushi, Takagi Ryosuke, Adachi Tomomi, Hoshino Tsutomu

    Symposium on the Chemistry of Natural Products, symposium papers   50 ( 0 )   517 - 522   2008

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    Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C_<35>-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of 1 was considerably greater at a higher temperature of 37℃ than at an optimal growth temperature of 28℃, while that of 2 remained unchanged at all the temperatures (20-40℃). A feeding experiment with D-[1-^<13>C] glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C_<35>-isoprenoid. The substrate 2-PP has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds has not been reported hitherto. Despite a careful search using the FASTA3 program, we could not detect any gene homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

    DOI: 10.24496/tennenyuki.50.0_517

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  • 4 Novel Halimane-type Diterpenes Produced by Pathogenic Mycobacterium tuberculosis : Molecular Cloning, Functional Analyses, Substrate Specificity and Biological Activity

    Nakano Chiaki, Okamura Tomoo, Sato Tsutomu, Hara Takashi, Dairi Tohru, Toyomasu Tomonobu, Sassa Takeshi, Hoshino Tsutomu

    Symposium on the Chemistry of Natural Products, symposium papers   48 ( 0 )   19 - 24   2006

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    Based upon the genomic database of Mycobacterium tuberculosis H37Rv, the gene Rv3377c,which is a putative terpene cyclase, was amplified by PCR and ligated into BamH I/Hind III site of pET 22b(+), and expressed in E. coli. BL21(DE3). The expressed Rv3377c gene product is almost insoluble (inclusion body), but coexpression of Rv3377c and chaperon in E. coli afforded the gene product as an active form. The functionally expressed gene product had an enzyme activity only for geranylgeranyl diphosphate (GGPP), but inert to geranyl-pp (GPP), farnesyl-pp (FPP), squalene and oxidosqulaene. The structural analysis of the enzymatic product revealed that this enzyme encodes the diterpene cyclase for producing the halimane diterpene skeleton. We named tuberculosinol 1 for the enzyme product. The detailed investigation on the gene product indicated that the actual product by the Rv3377c enzyme was tuberculosinol diphosphate 2. Cyclic diterpene skeletons are usually constructed after removing the PP functional group. After the trials to find the dephosphorylating enzyme, it has turned out that the flanking gene Rv3378c is responsible for the dephosphorylation reaction of 2, yielding the hydroxylated product (isotuberculosinol 3) and 1 resulting from a water attack. Both of the Rv3377c and Rv3378c have been found only the pathogenic species, but not in the non-pathogenic one. Recently, Russell and coworkers reported that the survival of Mycobacterium tuberculosis in the macrophage was attenuated by the gene disruption Thus, the two genes may be closely related to the pathogenicity. We evaluated the effect of 1 on phagocytic activity against zymosan, a yeast cell wall component that produces inflammation, using human macrophage-like U937 cells. Fluorescence microscopic analysis revealed that the phagocytosis of FITC-labeled zymosan by U937 cells treated with 10μM of 1 was reduced by about 50% compared to the cells without treatment. We discuss the detailed enzyme characterization including the kinetic data and substrate specificities. The substrate specificity of the Rv3378c enzyme was remarkably broad as well as cyc2, which was found in Streptomyces griseolosporeus. The Rv3378c and cyc2 accepted copalyl-PP (CDP), ent-CDP and syn-CDP as substrate, leading to the successful construction of a variety of diterpene skeletons.

    DOI: 10.24496/tennenyuki.48.0_19

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  • オキシドスクアレン-ラノステロール環化酵素欠損型の酵母変異株の取得

    SATO Tsutomu, HOSHINO Tsutomu

    Bulletin of the Faculty of Agriculture,Niigata University   56 ( 2 )   113 - 118   2004.3

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    我々はオキシドスクアレン-ラノステロール環化酵素(OLC)の触媒機構を解明するため、酵母の発現糸を構築することを計画した。しかし、野生型の酵母は自身のOLCを持っていることから、そのまま発現宿主として用いることはできない。したがって、 OLC欠損型の酵母変異株を取得することを目的に実験を行った。酵母OLC欠損型二倍体から胞子形成を経て一倍体を分離した。得られた株(3-5-1)について、抗生物質に対する抵抗性の調査や脂質成分の分析そしてOLCの酵素活性の検定を行った。全ての結果は、 3-5-1株がOLC欠損型一倍体であることを示唆していた。今後、 3-5-1株は外来OLCの発現宿主として利用することができると考えられる。In order to investigate the catalytic mechanism of oxidosqualene-lanosterol cyclase (OLC), we have planed to construct the expression systenl of OLC in yeast Saccharomyces cerevtsiae. However, wild-type yeast can not be used as the expression host of OLC from o山er organism, because yeasts naturally have its own OLC. Therefore, we made experiments to obtain the yeast OLC deficient mutant. The OLC deficient haploid was separated from the OLC deficient diploid after the sporulation. The obtained microorganism, named 3-5-1, was confirmed to be OLC deficient haploid by checking the resistance for antibiotics, analyzing the components of lipid and assaying the OLC activity. The strain, named 3-5-1, may be able to be used as the expression host of OLC from other organism in future.

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    Other Link: http://hdl.handle.net/10191/25024

  • Candida albicans由来のオキシドスクアレン-ラノステロール環化酵素の大腸菌における発現について

    SATO Tsutomu, HOSHINO Tsutomu

    Bulletin of the Faculty of Agriculture,Niigata University   56 ( 2 )   105 - 112   2004.3

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    In order to investigate the catalytic mechanism of oxidosqualene-lanosterol cyclase (OLC), we have tried to construct the overexpression system of OLC from C. albicans in E. colt. First, a natural type of OLC was expressed in E.coli. However, it was insoluble in E.coli cell and had no enzymatic activity. Therefore, nine kind of altered OLCs were constructed on the basis of successful methods for other enzymes to increase the solubility or the suggestion from our experimental results for the squalene-hopene cyclase: a fused OLC with Trx or Dsb, a OLC deleted N-terminal domain and mutant OLCs having squalene-hopene cyclase types of QW motif. However, all types of OLC were insoluble and had no enzymatic activity. These results suggested that the expression host of OLC must be eucaryotic cells such as yeast and insect.

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    Other Link: http://hdl.handle.net/10191/25023

  • スクアレン : ホペン環化酸素に保存されているフェニルアラニン残基とチロシン残基の触媒機能

    佐藤 努, 星野 力

    日本農芸化学会誌   77 ( 4 )   430 - 431   2003.4

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    DOI: 10.1271/nogeikagaku1924.77.430

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  • Squalene-hopene cyclase: catalytic mechanism and substrate recognition Reviewed

    T Hoshino, T Sato

    CHEMICAL COMMUNICATIONS   ( 4 )   291 - 301   2002

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    Rapid progress on the catalytic mechanism and substrate recognition by squalene-hopene cyclase, which has occurred only in the last several years, is reported. A series of site-directed mutation experiments and some squalene analogues have provided deep insight into the polycyclization mechanism and catalytic sites in conjunction with the information from X-ray crystal data.

    DOI: 10.1039/b108995c

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  • 34 Cyclization Mechanism of Squalene Proposed on the Basis of Site-directed Mutagenesis Experiments

    HOSHINO Tsutomu, SATO Tsutomu, Kouda Masanori, Ohhasi Shumi, Abe Takamasa

    Symposium on the Chemistry of Natural Products, symposium papers   41 ( 0 )   199 - 204   1999

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    Squalene cyclization mechanism and the active sites of the cyclase are discussed on the basis of the site-directed mutagenesis experiments. 1. DXDDTA motif: the initiation of the polycyclization and stabilization of carobocation intermediate of the initialy formed A-ring through the carboxylate anion of D377. 2. Phe365 stabilizes the C-8 carbocation intermediate of A/B-fused ring system through cation/π interaction. This interaction was confirmed by constructing the mutants F365Y and F365W; these mutants accelerated the reaction velocity and the significant lowering of activation energy for the polycyclization reaction. Tyr420 is also responsible for the formation of B-ring. 3. Phe 601is crucial for the construction of the 6-membered C/D-ring system. The mutant F601A produced the partially cyclized tricyclic 6/6/5-fused13,14 and tetracyclic 6/6/6/5-fused 18. These products could be formed by a Markovnikov closure and indicated the involvements of ring-expansion processes from the 5-membered into the 6-membered ring system for the construction of the C/D-ring system. Compound 18 was also isolated from the mutant W169F and W169H. The presumed carbocationic intermediates 12 and 17 was trapped by using the substrate analogues 27 and 32(34), respectively, with a high nucleophilic hydroxyl group, resulting in the formation of 31 and 35. Thus, we propose that the polycyclization mechanism proceeds via two ring-expansion steps for the formation of C- and D-rings, leading to the formation of anti-Markovnikov adduct 2. 4. The mutant I261A produced a series of carbocationic intermeidates 6/6/5-, 6/6/6/5-, and 6/6/6/6-fused ring systems, giving further insight into the polycycliation mechansim. In the enzymatic products, unnatural natural products are included. 5. Phe 605 would stabilize the cations of 17, 19 and 20 through cation-π interaction, as verified by the isolations of 6/6/6/5- and 6/6/6/6/5-ring skeletons. 6. The methyl at C10-posisiton of 1 is crucial to the correct folding in the active center, this being demonstrated by the substrate analouge 40. The proposed cyclization mechanism has been verified both by the kinetic measurments and by the isolation of the differently cyclized products resulting from each stage of the carbocationic intermediates. Alteration of the cyclase active sites afforded multiple triterpenes in addtion to the understanding of the fundamental issues of squalene cyclization mechanism, suggesting the possibility that we will be able to generate novel triterpenes (unnatural natural products) by rational genetic engineering of squalene cyclase.

    DOI: 10.24496/tennenyuki.41.0_199

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  • 25 Substrate Recognition of Squalene and Oxidosqualene Cyclases : Substrate Conformations are Controlled by Molecular Recognition of the Cyclase Enzymes toward Methyl Groups of the Substrates?

    HOSHINO Tsutomu, SATO Tsutomu, KONDO Tomohiro, SAKAI Yoshiyuki, ISHIBASHI Eiichi

    Symposium on the Chemistry of Natural Products, symposium papers   39 ( 0 )   145 - 150   1997

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    There has been remarkable advances in the studies of oxidosqualene and squalene cyclases in the past ten years. First, complete purifications of the enzymes have been attained from several species such as vertebrates, plant, yeast and bacterium, irrespective of unstable and membrane-bound nature. Second, application of cDNA cloning technique to the cyclases have succeeded in determining the alignments of amino acids from various biological sources including human. Third, the substrate analogues including the suicide inhibitors have made a great contribution to the polycyclization mechanism. In this symposium, we report a definitive evidence that the polycyclization proceeds via the discrete carbocationic intermediate formed during the cyclization, but not via a concerted manner as proposed before; substitution of methyls with ethyl groups at 10- and 15-positions 6 halted the enzymic reaction at the monocyclic stage 7 and 8. Substrate analogue 9 with ethyl group at 15-position afforded 10 (normal cyclization) and 11 (tricyclic 6/6/5), the latter being formed under the control of Markovnikov rule. This is in contrast to the protosterol cation, which is formed under anti-Markovnikov control. Thus, oxidosqualene would be converted via the 5-membered intermediate 12 under the control of Markovnikov rule, which then undergoes a ring expansion to form 6-membered C-ring. Mechanisms of the formation of tetrahymanol and hopene from squalene itself are discussed, especially on the terminal cyclization (E-ring formation). Substrate analogs 14, 15, 18, 22 have demonstrated the cyclization mechanisms as follows: for tetrahymanol cyclase, the terminal cyclization proceeds by the process of stereoelectronic control, suggesting little participation of the enzyme, while hopene cyclase strongly binds to the terminal methyl groups to form 5-membered E-ring. From our studies, methyl groups of the substrate seem to bind the cyclase enzymes and the substrates were then subjected to the folding of chair-boat or chair-chair inside the enzymes leading to production of the desired triterpene skeletons. Over-expression of hopene cyclase was achieved successfully and the point mutations of amino acids in QW motif proved that the functions of D and W are crucial for the enzyme activity.

    DOI: 10.24496/tennenyuki.39.0_145

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  • 好熱好酸菌Alicyclobacillus acidocaldarius由来スクアレン-ホペン閉環酵素(SHC)の大腸菌における高発現系の構築 : 有機化学・天然物化学

    佐藤 努, 金井 芳則, 星野 力

    日本農藝化學會誌   70 ( 0 )   241 - 241   1996.3

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  • 非天然型オノセロイドの酵素合成

    平井 奈実, 井上真緒, 上田 大次郎, 品田哲郎, 佐藤努

    香料・テルペンおよび精油化学に関する討論会  2020.10 

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  • 非末端環化型トリテルペン/セスクアテルペン環化酵素のゲノムマイニング

    寒河江侑加, 井上真緒, 上田大次郎, 佐藤努

    香料・テルペンおよび精油化学に関する討論会  2020.10 

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  • 龍涎香人工合成経路の構築:アンブレインと香気成分の効率的合成と破骨細胞分化誘導活性

    山辺 陽太, 川越 幸奈, 奧野琴音, 上田大次郎, 柿原 嘉人, 佐藤努

    2020.3 

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  • Mycobacterium属細菌由来新型セスクアテルペン環化酵素の高感度活性測定法の構築とそれを利用した探索

    阿部透, 尾崎真夢, 吉田優里, 三浦彩奈, 相良昌寛, 上田大二郎, 金古堅太郎, 三ツ井敏明, 佐藤努

    日本農芸化学会  2019.3 

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  • スクレイレン‐アンブレイン環化酵素の創出:アンブレインはスクレンレンから二つの経絡を経由して一つの酵素により合成される

    山辺陽太, 奥野琴音, 井上真緒, 上田大次郎, 佐藤努

    日本蛋白質科学会年会プログラム・要旨集  2018.5 

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  • スクアレン‐アンブレイン環化酵素の創出:アンブレインはスクアレンから2つの経路を通して1つの酵素によって合成できる

    上田大次郎, 奥野琴音, 星野力, 佐藤努

    日本生物工学会大会講演要旨集  2017.8 

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  • スクアレン‐アンブレイン環化酵素の創出:アンブレインはスクアレンから一つの酵素によって2つの経路を経て合成できる

    奥野琴音, 上田大次郎, 村上瑞気, 星野力, 佐藤努

    イソプレノイド研究会例会講演要旨集  2016.9 

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  • スクアレンからアンブレインへの一段階酵素合成

    奥野琴音, 上田大次郎, 星野力, 佐藤努

    日本農芸化学会大会講演要旨集(Web)  2016.3 

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  • 龍涎香の主成分アンブレインの酵素合成

    佐藤努

    日本農芸化学会大会講演要旨集(Web)  2016.3 

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  • 3P-269 Biosynthesis of rare terpenoids in Bacillus clausii

    Ueda Daijiro, Yamaga Hiroaki, Murakami Mizuki, Totsuka Yusuke, Shinada Tetsuro, Sato Tsutomu

    日本生物工学会大会講演要旨集  2015 

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  • 龍涎香の主成分アンブレインの酵素合成

    上田大次郎, 星野力, 佐藤努

    香料・テルペンおよび精油化学に関する討論会講演要旨集  2014.9 

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  • スクアレンの両末端環化:オノセロイド合成酵素の初めての同定および龍涎香主成分ambreinの酵素合成

    上田大次郎, 星野力, 佐藤努

    イソプレノイド研究会例会講演要旨集  2014.9 

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  • スクアレンの両末端環化:オノセロイド合成酵素の初めての同定および龍涎香主成分ambreinの酵素合成

    上田大次郎, 星野力, 佐藤努

    酵素工学研究会講演会講演要旨集  2014.4 

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  • スクアレンの両末端環化:オノセロイド合成酵素の初めての同定および龍涎香主成分ambreinの酵素合成

    上田大次郎, 星野力, 佐藤努

    日本農芸化学会大会講演要旨集(Web)  2014.3 

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  • Novel and unnatural terpenes found from sesquarterpene cyclase study

    Ueda Daijiro, Yamaga Hiroaki, Okamoto Wataru, Totsuka Yusuke, Shinada Tetsuro, Hoshino Tsutomu, Sato Tsutomu

    Symposium on the Chemistry of Natural Products, symposium papers  2014 

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    <p>Biosynthetic studies on the sesquarterpenes, a family of C<sub>35 </sub>terpenes, in Bacillus subtilis have identified a new type of terpene cyclase, tetraprenyl-b-curcumene synthase (TS), which lacks sequence homology to any known terpene synthases. We recently revealed that Bacillus megaterium tetraprenyl-b-curcumene cyclase (TC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail C<sub>35</sub> 1 and tail-to-tail C<sub>30</sub> 3 into pentacyclic 2 and bicyclic 4, respectively, in vivo (Scheme 1). In this presentation, we report novel and unnatural terpenes found from sesquarterpene cyclase (TS and TC) study.</p><p>1) We discovered head-to-tail types of novel acyclic sesterterpene 5 and triterpene 6 in B. clausii, and revealed that compounds 5 and 6 are biosynthesized by TS homolog (Bcl-TS) from GFPP and HexPP, respectively (Scheme 2). The present study revealed that TS homologs are a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. Genome mining of this new terpene synthase family will lead to discoveries of novel terpene synthases and terpenoids in the future.</p><p>2) The enzymatic cyclization of head-to-tail acyclic triterpene 6isolated from Bacillus clausii using B. subtilis TC resulted in the formation of two unnatural pentacyclic triterpenes 7and 8 (Scheme 3). It was revealed that B. subtilis TC, which forms tetracyclic terpenoid scaffold from tetraprenyl-β-curcumene in vivo, could be used to construct the 6/6/6/6/6-fused pentacyclic scaffold in vitro, suggesting that the active site cavity of TC has sufficient space to accommodate this unnatural pentacyclic scaffold. </p><p>3) We revealed that TC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase (Scheme 4). Also, we report the first onoceroids from bacterial origin. Our discoveries suggested that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of TC enabled the synthesis of (+)-ambrein 11, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase–catalyzed reaction from easily available squalene (Scheme 5).</p>

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  • 17 Sesquarterpenes (C_<35> Terpenes) from Bacteria : Identifications of Unique Biosynthetic Enzymes and Search for Novel Natural Products(Oral Presentation)

    Sato Tsutomu, Nakajima Mami, Hoshino Tsutomu, Hoshino Hiroko, Yoshida Satoru, Takizawa Kazuo, Orito Yuriko, Takagi Ryosuke, Tanno Mizuki, Kudo Hanayo, Ono Eri

    Symposium on the Chemistry of Natural Products, symposium papers  2011 

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    In this study, mono- and pentacyclic C_<35> terpenes from Bacillus subtilis were biosynthesized via the cyclization of C_<35> isoprenoid by using purified enzymes, including the first identified new terpene cyclase, tetrapreny1-13-curcumene synthase, that shows no sequence homology to any of the known terpene cyclases. Based on these findings, we propose that these C_<35> terpenes are called the new family of "sesquarterpenes." This study demonstrated that a tetrapreny1-β-curcumene cyclase (TC) from B. subtilis, which was originally identified as a sesquarterpene cyclase converting a head-to-tail type of monocycle to a pentacycle, also cyclized a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-Hydroxypolypoda-13,17,21-triene. The 8α-Hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium, suggesting that the TC is bifunctional, cyclizing both tetrapreny1-α-curcumene and squalene in vivo. This is the first report describing the bifunctional terpene cyclase, which biosynthesizes 2 classes of cyclic terpenes with different numbers of carbons as natural products in the organism. Non-pathogenic Mycobacterium species also produce cyclic sesquarterpenes, which are biosynthesized via cyclization of Z-type C_<35> polyprenyl diphosphate. To provide deeper insight into the biosynthesis of sesquarterpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M vanbaalenii identified by genomic analysis. Mvan_3822, a novel bi-functional Z-prenyltransferase, biosynthesizes C_<35>-heptaprenyl diphosphate as a main product from E,E-FPP and E,E,E-GGPP, but produces a C_<50>-decaprenyl diphosphate from GPP. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C_<35>-terpenes, 14E- and 14Z-dehydroheptaprenylcycline, were identified as minor metabolites in non-pathogenic Mycobacterium cells. Sesquarterpenes could be biosynthesized by two routes, in which E- and Z-geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of sesquarterpenes may work to reduce both E- and Z-prenyl residues. The studies on sesquarterpenes promise to be an attractive field for expanding our understanding of the terpene world.

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  • P-85 Biosynthesis of a novel cyclic C_<35>-terpene by the cyclisation of a Z-type C_<35>-polyprenyl diphosphate obtained from a nonpathogenic Mycobacterium species(Poster Presentation)

    Sato Tsutomu, Kigawa Atsushi, Takagi Ryosuke, Adachi Tomomi, Hoshino Tsutomu

    Symposium on the Chemistry of Natural Products, symposium papers  2008 

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    Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C_<35>-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of 1 was considerably greater at a higher temperature of 37℃ than at an optimal growth temperature of 28℃, while that of 2 remained unchanged at all the temperatures (20-40℃). A feeding experiment with D-[1-^<13>C] glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C_<35>-isoprenoid. The substrate 2-PP has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds has not been reported hitherto. Despite a careful search using the FASTA3 program, we could not detect any gene homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

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Awards

  • 第16回酵素応用シンポジウム研究奨励賞

    2015.6  

    佐藤 努

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  • 農芸化学奨励賞

    2012.3  

    佐藤 努

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  • Biosci. Biotechnol. Biochem.論文賞

    2002.3  

    佐藤 努

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Research Projects

  • 活性酸素の非酵素反応が関与するテルペノイド系天然物生合成

    Grant number:19K22273

    2019.6 - 2022.3

    System name:科学研究費助成事業 挑戦的研究(萌芽)

    Research category:挑戦的研究(萌芽)

    Awarding organization:日本学術振興会

    佐藤 努, 岡田 正康, 須原 義智, 廣田 佳久

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    Grant amount:\6500000 ( Direct Cost: \5000000 、 Indirect Cost:\1500000 )

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  • Neural basis for inhibitory effects of exercises on enhanced nociception in the orofacial region under psychological stress conditions

    Grant number:19K10353

    2019.4 - 2022.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    Okamoto Keiichiro

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    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    Stress controls are critical to alleviate stress-induced pain in human. However, 亜as we cannot avoid stressful events in our life, it could be appropriate to control adverse stress responses by means of convenient ways, like exercise. This study determined the effect of daily treadmill running on masseter muscle nociception under psychological stress conditions. The results indicated that enhanced masseter muscle nociception indicated by orofacial nocifensive behaviors and neural activities (c-Fos and FosB) in the upper cervical dorsal horn were inhibited by daily treadmill running exercise.These findings supported our hypothesis that daily exercise could have therapeutic and preventive roles on deep craniofacial nociception associated with psychological stress conditions.

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  • 対称分子から非対称環状骨格へのトリテルペン生合成マシナリーの解析とリデザイン

    Grant number:19H04648

    2019.4 - 2021.3

    System name:科学研究費助成事業 新学術領域研究(研究領域提案型)

    Research category:新学術領域研究(研究領域提案型)

    Awarding organization:日本学術振興会

    佐藤 努

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    Grant amount:\7540000 ( Direct Cost: \5800000 、 Indirect Cost:\1740000 )

    新しい酵素である「アンブレイン合成酵素」を人工的に創出し、生合成による供給経路の確立に着手するとともに、アンブレインの香気成分への化学変換、ならびに薬理活性評価研究をあわせて展開した。
    まず、細菌の酵素をリデザインすることにより、アンブレインを大量生産するための「アンブレイン合成酵素」を創出した。アンブレインの収率は、以前報告されたものの約20倍向上できた。
    次に、アンブレインから龍涎香の香気成分への効率的な化学変換系を確立した。香気成分の収率は、既知の方法や天然の龍涎香の約8~15倍であった。光増感剤の種類によって、香り成分の比率が異なっており、反応条件によって「人工龍涎香」の香りを変えることができることが示唆された。
    最後に、酵素合成したアンブレインを用いて、破骨細胞の分化促進とアミロイドβ誘導性神経細胞死の抑制という2つの新しい薬理活性を見出した。今後、骨代謝改善とアルツハイマー病予防に働く薬剤の開発につながる可能性がある。生合成経路が不明な希少天然物、アンブレインを研究室内で創出した酵素を使った人工生合成経路で合成し、その合成効率を飛躍的に高めることができた。アンブレインの生合成においては、真の生産者のみならず天然由来の生合成酵素も未だ特定されていない。本研究は、生合成遺伝子の利用の適用が困難な天然物を異なる生合成酵素のリデザインから創出したことに大きな意義を持つと考えられる。

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  • Discovery of new terpene cyclization pathway and identification of various novel terpenes

    Grant number:18H02145

    2018.4 - 2021.3

    System name:Grants-in-Aid for Scientific Research

    Research category:Grant-in-Aid for Scientific Research (B)

    Awarding organization:Japan Society for the Promotion of Science

    Sato Tsutomu

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    Grant amount:\17680000 ( Direct Cost: \13600000 、 Indirect Cost:\4080000 )

    We were able to identify isoprenyl diphosphate synthases and prenyl reductase of Mycobacterium spp. Using these enzymes, we succeeded in preparing a 14C high-sensitivity substrate for the identification of Z-type sesquarterpene cyclase that contributes to the "pathway for cyclizing Z-type isoprenoids". Using this system, the native enzyme was partially purified by various chromatographies, and ZTC candidates could be narrowed down by LC-MS/MS. We also succeeded in X-ray structural analysis of the homologue of the triterpene / sesquarterpene cyclase that contributes to the "pathway that initiates cyclization from the non-terminal". The modeling structure suggested that there was a space where the substrate ends could bind near the initiation reaction site.

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  • Elucidation and utilization of biosynthetic pathway not involving known isopentenyl diphosphate isomerase homologs

    Grant number:16K14911

    2016.4 - 2019.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Research category:Grant-in-Aid for Challenging Exploratory Research

    Awarding organization:Japan Society for the Promotion of Science

    Sato Tsutomu, KUZUYAMA Tomohisa, UMENO Daisuke

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    Grant amount:\3640000 ( Direct Cost: \2800000 、 Indirect Cost:\840000 )

    Farnesyl diphosphate synthetase synthesized farnesyl diphosphate using only isopentenyl diphosphate as a substrate. This result suggested that it also has isopentenyl diphosphate isomerase activity. That is, it turned out that it is a bifunctional enzyme. Currently, we are conducting research aimed at elucidating the catalytic mechanism.

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  • Creation of novel terpenoids from biosynthetic study on sesquarterpene

    Grant number:25450149

    2013.4 - 2016.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    Sato Tsutomu, FUJIHASHI Masahiro, SHINADA Tetsuro

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    Grant amount:\5200000 ( Direct Cost: \4000000 、 Indirect Cost:\1200000 )

    Genome mining and analysis of multifunction of enzyme found novel terpenes. Onoceroid synthase was first identified. In addition, enzymatic synthesis of ambrein was succeeded. Search for novel type of terpene synthase from Mycobacterium and analysis of catalytic mechanism of novel enzyme were also steadily performed.

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  • 新規酵素群の探索を軸とするイソプレノイド分子多様性の拡充

    Grant number:25108709

    2013.4 - 2015.3

    System name:科学研究費助成事業 新学術領域研究(研究領域提案型)

    Research category:新学術領域研究(研究領域提案型)

    Awarding organization:日本学術振興会

    佐藤 努

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    Grant amount:\7020000 ( Direct Cost: \5400000 、 Indirect Cost:\1620000 )

    前年度、Bacillus属細菌からファルネシルファルネシルアセトンを天然物として初めて発見した。本年度、新型酵素の可能性があるファルネシルファルネシルアセトン合成酵素を探索する手始めとして、Bacillus属細菌の無細胞抽出液を用いて想定基質のメナキノンー7と様々な条件で反応実験を行ったが、ファルネシルファルネシルアセトン生成活性を見いだすことができなかった。一方、活性酸素による非酵素的反応によって特異的にメナキノンー7から生産されることをin vitro実験で明らかになった。天然物として知られる他のポリプレニルアセトンも同様の機構によって生合成されている可能性があると考えている。

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  • Elucidation of catalytic mechanism of two characteristic enzymes and its application based on crystal structures.

    Grant number:24570130

    2012.4 - 2015.3

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    FUJIHASHI Masahiro, SATO Tsutomu, ISHIDA Toyokazu

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    Grant amount:\5460000 ( Direct Cost: \4200000 、 Indirect Cost:\1260000 )

    This project targeted two enzymes. Regarding the first enzyme ODCase, we determined about 10 crystal structures using various mutants and/or ligands. The best resolution of the determined structures is 1.03 A, which is much better than those of the over 200 determined ODCase structures thus far. Based on the structures with biochemical and computational analyses, we elucidated that substrate distortion contributes to 10-15% free energy decrease of the transition state by ODCase. Regarding the other enzyme Cyc2, we elucidated the enzyme is not appropriate for the crystallographic analysis. We are now attempt to purify and crystallize a homologous protein from a different organism.

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  • メタゲノム由来モジュールライブラリーを用いた非天然型ペプチドの創出

    Grant number:23108530

    2011.4 - 2013.3

    System name:科学研究費助成事業 新学術領域研究(研究領域提案型)

    Research category:新学術領域研究(研究領域提案型)

    Awarding organization:日本学術振興会

    佐藤 努

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    Grant amount:\3510000 ( Direct Cost: \2700000 、 Indirect Cost:\810000 )

    チコリンの生合成に寄与する22個のモジュールからなる非リボソーム型ペプチド合成酵素の全塩基配列(約73 kb)を決定できた。ゲノム内に非常に類似した配列が多数存在することから、紆余曲折があったが、次世代シーケンサー解析、BACクローニング、PCR等によって達成することができた。チコリンの構成アミノ酸のDLは一部決定できていなかったが、Cドメインの配列情報から、立体化学を予想することができた。現在、チコリン生合成遺伝子の大腸菌への導入を試みている。
    チコリン生合成遺伝子の全長配列決定に手間取っていたため、枯草菌由来のサーファクチン生合成も新たにターゲットに加えた。枯草菌でモジュール置換NRPSライブラリーを作るため、サーファクチンの2番目のモジュール遺伝子を破壊した枯草菌の作製や、導入するプラスミドの構築を達成した。したがって、枯草菌由来サーファクチン生合成遺伝子発現系を構築できた。
    汚泥、温泉、水田、堆肥などの土壌から抽出したメタゲノムを鋳型として、数種の組み合わせの保存アミノ酸配列の縮重プライマーを用いてPCRした。ある組み合わせにおいてNRPSと推定されるサイズのバンドが確認できた。プラスミドに導入後、数個のクローンのDNA配列を確認したところ、ほとんどがNRPS遺伝子であり、かつ配列には多様性があることを確認できた。現在、メタゲノムからモジュールライブラリーを作製し、枯草菌由来サーファクチン生合成遺伝子発現系に導入し、非天然型ペプチドの創出を行っている。

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  • Search for novel C35 terpene cyclases, genome mining and biosynthesis of unnatural natural products

    Grant number:23780114

    2011 - 2012

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

    Research category:Grant-in-Aid for Young Scientists (B)

    Awarding organization:Japan Society for the Promotion of Science

    SATO Tsutomu

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    Grant amount:\4550000 ( Direct Cost: \3500000 、 Indirect Cost:\1050000 )

    We found novel terpen cyclase from Bacillus subtilis, and proposed that C35 terpene should be called sesquarterpene. We identified a novel acyclic sesterterpene/triterpene synthase from Bacillus clausii. We found first bifunctional terpene cyclase which cyclizes two class of terpenes in vivo. We also performed isolation of a novel sesquarterpene from Mycobacterium and biosynthesis of unnatural terpenoids.

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  • Search for novel families of terpene cyclases from bacteria and analyses of physiological functions and bioactivities of C35 terpenes

    Grant number:21780109

    2009 - 2010

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

    Research category:Grant-in-Aid for Young Scientists (B)

    Awarding organization:Japan Society for the Promotion of Science

    SATO Tsutomu

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    Grant amount:\4550000 ( Direct Cost: \3500000 、 Indirect Cost:\1050000 )

    A bifunctional Z-prenyltransferase that preferentially synthesize C_<35> or C_<50> products was found from the study on the mycobacterial C_<35> terpenes. The structure of two novel C_<35> terpenes suggested that a prenyl reductase reducing both E and Z prenyl residues was responsible for the biosynthesis of C_<35> terpenes. On the other hand, a novel family of terpene cyclases and tetraprenyl-β-curcumene cyclase from Bacillus subtilis could be identified by the in vitro enzymatic reaction.

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  • Search and biosynthesis of novel cyclic C35-terpenes produced by Mycobacterium

    Grant number:19780085

    2007 - 2008

    System name:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

    Research category:Grant-in-Aid for Young Scientists (B)

    Awarding organization:Japan Society for the Promotion of Science

    SATO Tsutomu

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    Grant amount:\3750000 ( Direct Cost: \3300000 、 Indirect Cost:\450000 )

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  • 潜在する微生物テルペノイドの発掘及び新型テルペン環化酵素遺伝子の取得

    Grant number:17780088

    2005 - 2006

    System name:科学研究費助成事業 若手研究(B)

    Research category:若手研究(B)

    Awarding organization:日本学術振興会

    佐藤 努

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    Grant amount:\3600000 ( Direct Cost: \3600000 )

    生合成研究:Mycobacterium chlorophenolicumから単離されたC_<35>環状テルペンはZ型のC_<35>直鎖状ポリプレニルニリン酸の環化反応によって生合成されると予想している。それを支持する証拠を得るため、市販の安価な試薬から容易に合成できる短鎖のC_<10>とC_<15>のポリプレニルニリン酸を基質として用いて無細胞抽出液と反応させた。E型のC_<10>ポリプレニルニリン酸は反応しなかったのに対し、Z型はC_<35>環状テルペンの部分構造と類似したリモネンを生成した。また、ω.E.E体のC_<15>のポリプレニルニリン酸も反応せず、ω, Z, Zで体は環化反応した。これらの結果は、Z型のポリプレニルニリン酸にのみ基質特異性をもつテルペン環化酵素が存在することを示していた。現在知られているテルペン環化酵素はE型を天然基質とするものだけであることから、新規性の高いテルペン環化酵素が存在していることが示唆された。
    遺伝子クローニング:昨年度までに2つのZ型プレニルトランスフェラーゼ遺伝子の一部を縮重プライマーによってクローニングしていた。今年度、染色体歩行によって上・下流の塩基配列を読み進んだ。高いGC含量の影響等により容易に読める配列ではなかったが、様々なシーケンス反応の条件を検討し改良した結果、Z型プレニルトランスフェラーゼ遺伝子の全長を読むことができた.現在、更に上・下流を読むことによってC_<35>環状テルペン生合成酵素の候補遺伝子を探している.また、2つのZ型プレニルトランスフェラーゼの生成物を同定するため、pET系ベクターで発現させる実験を進行した。

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  • 新規テルペン環化酵素の微生物ゲノムからの発掘

    Grant number:15780083

    2003 - 2004

    System name:科学研究費助成事業 若手研究(B)

    Research category:若手研究(B)

    Awarding organization:日本学術振興会

    佐藤 努

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    Grant amount:\3600000 ( Direct Cost: \3600000 )

    本研究では、ゲノム解析から見出された推定のテルペン環化酵素の機能解析を行い、新規環状骨格を形成する環化酵素の発見につなげることを目的としている。
    本年度において、すでにクローニングと大腸菌における発現を達成している結核菌Mycobacterium tuberculosis由来の推定環化酵素について研究を進めた。基質はゲラニルゲラニル2リン酸であることが判明していたが、生成物の構造は確定していなかった。そこで、単離・精製し、MS及びNMRによって構造解析したところ、今までに見出されていないハリマン骨格のジテルペン(二リン酸体)であることが解った。
    他のミコバクテリア属にも新規テルペンが存在するのではないかと予想し、Mycobacterium smegmatisの炭化水素成分のGC-MS分析を行ってみた。その結果、新規物質の可能性がある2種類の脂質を見出すことができた。大量培養後、各種クロマトグラフィーによって単離・精製した。MS及びNMRによって構造解析したところ、両者ともC_<35>の単環性の炭化水素であることが判明した。構造からヘプタプレニル二リン酸の二リン酸脱離から開始する環化反応によって生合成されることが推測される。我々の知る限り、このようなC_<35>テルペン類の報告はなく、天然物として初めての例ではないかと考えている。
    スクアレン環化酵素の研究も同時に進めた。変異型酵素を機能解析し、反応最終段階の脱プロトン化の触媒機構について新たな知見を得ることができた。また、原核生物のスクアレン環化酵素を真核生物由来トリテルペン環化酵素型(Gly600欠損型酵素)の基質特異性へ改変することに成功した。

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  • スクアレン環化酵素の触媒反応機構の分子生物学的手法による解明

    Grant number:00J05448

    2000 - 2001

    System name:科学研究費助成事業 特別研究員奨励費

    Research category:特別研究員奨励費

    Awarding organization:日本学術振興会

    佐藤 努

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    Grant amount:\2400000 ( Direct Cost: \2400000 )

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Teaching Experience

  • 応用生命科学セミナー

    2023
    Institution name:新潟大学

  • 香粧品科学

    2023
    Institution name:新潟大学

  • 機器分析学

    2023
    Institution name:新潟大学

  • スタディ・スキルズAIc

    2022
    Institution name:新潟大学

  • Topics in Biotechnology and Biochemistry

    2022
    Institution name:新潟大学

  • スタディ・スキルズAIIc

    2022
    Institution name:新潟大学

  • 自然科学総論IV

    2022
    Institution name:新潟大学

  • スタディ・スキルズAIb

    2021
    Institution name:新潟大学

  • スタディ・スキルズAIIb

    2021
    Institution name:新潟大学

  • 応用生命・食品科学セミナーII

    2021
    Institution name:新潟大学

  • 応用生命・食品科学概論

    2020
    -
    2021
    Institution name:新潟大学

  • 応用生命・食品科学特論

    2020
    Institution name:新潟大学

  • 生物有機化学

    2019
    Institution name:新潟大学

  • 有機化学実験(農)

    2019
    Institution name:新潟大学

  • 農学入門Ⅱ

    2019
    Institution name:新潟大学

  • 農学入門Ⅰ

    2019
    Institution name:新潟大学

  • 生命を知る

    2018
    Institution name:新潟大学

  • 有機化学(農)

    2018
    Institution name:新潟大学

  • Topics in Biotechnology and Biochemistry

    2018
    -
    2022
    Institution name:新潟大学

  • 基礎化学

    2017
    Institution name:新潟大学

  • 応用生物化学科インターンシップ

    2015
    -
    2018
    Institution name:新潟大学

  • 応用生物化学概論

    2015
    -
    2018
    Institution name:新潟大学

  • 科学英語演習

    2015
    -
    2018
    Institution name:新潟大学

  • 生命・食料科学セミナーAⅠ

    2015
    Institution name:新潟大学

  • 生命・食料科学特定研究AⅠ

    2015
    Institution name:新潟大学

  • 研究発表演習(中間発表)

    2012
    -
    2015
    Institution name:新潟大学

  • 応用生命・食品科学演習(学会発表)

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅠ

    2012
    -
    2015
    Institution name:新潟大学

  • 文献詳読Ⅰ

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅡ

    2012
    -
    2015
    Institution name:新潟大学

  • 文献詳読Ⅱ

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅠ

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅡ

    2012
    -
    2015
    Institution name:新潟大学

  • 分子生命科学演習Ⅰ

    2011
    -
    2019
    Institution name:新潟大学

  • 分子生命科学演習Ⅱ

    2011
    -
    2018
    Institution name:新潟大学

  • 生命と環境の化学 I

    2011
    -
    2016
    Institution name:新潟大学

  • 分子生命科学実験

    2010
    -
    2018
    Institution name:新潟大学

  • 機器分析化学Ⅱ

    2010
    -
    2017
    Institution name:新潟大学

  • 自然科学総論Ⅲ

    2009
    Institution name:新潟大学

  • 生体物質構造解析学

    2008
    Institution name:新潟大学

  • 微生物天然物化学

    2007
    Institution name:新潟大学

  • 微生物機能化学

    2007
    -
    2022
    Institution name:新潟大学

  • 生物機能物質化学

    2007
    -
    2018
    Institution name:新潟大学

  • 有機化学実験

    2007
    -
    2018
    Institution name:新潟大学

  • 化学

    2007
    -
    2015
    Institution name:新潟大学

  • 機器分析化学

    2007
    -
    2008
    Institution name:新潟大学

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