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Toshiyuki Waki

Section

Graduate School of Engineering

Job title

Assistant Professor

Degree

博士（工学）（東北大学）
修士（工学）（東北大学）

researchmap

https://researchmap.jp/7000020943

J-GLOBAL ID

201701009178051715

Research History 1

2016/10 - Present

Tohoku University　Graduate School of Engineering Department of Biomolecular Engineering

Professional Memberships 3

日本植物バイオテクノロジー学会
JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY
JAPANESE SOCIETY FOR PLANT CELL AND MOLECULAR BIOLOGY

Research Interests 4

カルコン合成酵素
タンパク質間相互作用
フラボノイド
植物特化代謝

Research Areas 2

Life sciences / Molecular biology /
Life sciences / Applied molecular and cellular biology /

Awards 2

2018年度日本農芸化学会東北支部若手奨励賞

2018/09　公益社団法人日本農芸化学会東北支部　フラボノイド生合成における多酵素複合体（メタボロン）に関する研究
The JSPCMB Student Award

2016/09

Papers 25

Suppression of fecal phenol production by oral supplementation of sesamol: inhibition of tyrosine phenol-lyase by sesamol. International-journal

Daiki Oikawa, Zion Byun, Bunzo Mikami, Aina Gotoh, Toshihiko Katoh, Ryo Ueno, Aruto Nakajima, Satoshi Yamashita, Wakako Ikeda-Ohtsubo, Seiji Takahashi, Toshiyuki Waki, Koichi Kikuchi, Takaaki Abe, Takane Katayama, Toru Nakayama

Food & function　2025/04/15

DOI： 10.1039/d4fo04839c 　

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Phenol is produced from dietary L-tyrosine by the action of tyrosine phenol-lyase (TPL) of gut bacteria and contributes to various physiological disorders, including skin diseases, certain cancers, and kidney dysfunction. We found that oral supplementation of sesamol (36 or 180 μg mL-1) ad libitum for 14 days in mice significantly suppressed fecal phenol production. Fecal microbiota structure analysis in sesamol-supplemented and control groups revealed that their overall microbiota structures were indistinguishable. To explain the sesamol-induced suppression of fecal phenol production, we characterized inhibition of bacterial TPL by sesamol in vitro. Sesamol specifically inhibited bacterial TPL in a mixed-type fashion (Ki, 135 μM), which was rationalized by computational docking studies using the crystal structure of Pantoea agglomerans TPL that was determined at 1.3 Å resolution. Sesamol was detected at 0-0.295 μmol g-1 feces in the sesamol-supplemented group. Given the Ki value of sesamol for TPL inhibition, these levels may not have been sufficient to fully inhibit TPL and suppress fecal phenol production. Therefore, the observed suppression of fecal phenol production upon oral sesamol supplementation arose not solely from the inhibition of TPL by sesamol, but also potentially from the effects of metabolites derived from sesamol and the antioxidant activities of sesamol and related metabolites. Nevertheless, these findings highlight the potential for using sesamol to prevent physiological disorders associated with phenol production by the gut microbiota.
Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles

Seiji Takahashi, Miki Suenaga-Hiromori, Tomoki Ishii, Nadia Nur Shazana Binti Abu Talib Khan, Tomoyo Mikami, Tomohiro Takahashi, Chiho Minakawa, Fumihiro Yanbe, Toshiyuki Waki, Toru Nakayama, Riki Imaizumi, Taro Yanai, Kunishige Kataoka, Satoshi Yamashita, Kohei Takeshita, Hiroaki Matsuura, Naoki Sakai, Masaki Yamamoto, Haruhiko Yamaguchi, Yukino Miyagi-Inoue, Kazuhisa Fushihara, Yuzuru Tozawa

2024/07/03
Publisher: Springer Science and Business Media LLC
DOI： 10.21203/rs.3.rs-3615345/v1 　

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Abstract <p>Natural rubber (NR) is a sustainable biopolymer consisting mainly of cis-1,4-polyisoprene. Modifying an NR biosynthetic enzyme is a promising strategy to bioproduce novel polymers. Here, we have elucidated the NR biosynthetic mechanism and successfully developed novel enzymes that synthesise NR-sized polyisoprenes with unnatural substrates. NR is synthesised by a cis-prenyltransferase (cPT) on rubber particles (RPs), NR-harbouring lipid monolayer membrane organelles. However, the key to NR biosynthesis is not specialised cPTs, but the proper arrangement of cPTs on RPs since cPTs from various non-NR-producing organisms, such as humans, synthesise NR when introduced into the RPs. A tomato cPT, which condenses only one isoprene unit, was engineered to synthesise novel NR-sized polyisoprenes with artificial substrates by modifying residues for product size determination. Furthermore, the introduction of a modified trans-prenyltransferase into RPs led to the synthesis of NR-sized trans-1,4-polyisoprenes. This RP system could be used as a versatile platform for enzymatic polyisoprenoid synthesis.</p>
Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

Kazuhiro Ohara, Yasuhiro Oshima, Hideaki Unno, Satoru Nagano, Masami Kusunoki, Seiji Takahashi, Toshiyuki Waki, Satoshi Yamashita, Toru Nakayama

Journal of bioscience and bioengineering　2024/06/24

DOI： 10.1016/j.jbiosc.2024.05.010 　

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SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.
Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity

Toshiyuki Waki, Riki Imaizumi, Kaichi Uno, Yamato Doi, Misato Tsunashima, Sayumi Yamada, Ryo Mameda, Shun Nakata, Taro Yanai, Kohei Takeshita, Naoki Sakai, Kunishige Kataoka, Masaki Yamamoto, Seiji Takahashi, Toru Nakayama, Satoshi Yamashita

Biochemical and Biophysical Research Communications　150080-150080　2024/05
Publisher: Elsevier BV
DOI： 10.1016/j.bbrc.2024.150080 　

ISSN： 0006-291X
Structural‐Functional Correlations between Unique N‐terminal Region and C‐terminal Conserved Motif in Short‐chain cis‐Prenyltransferase from Tomato

Riki Imaizumi, Hiroaki Matsuura, Taro Yanai, Kohei Takeshita, Shuto Misawa, Haruhiko Yamaguchi, Naoki Sakai, Yukino Miyagi‐Inoue, Miki Suenaga‐Hiromori, Toshiyuki Waki, Kunishige Kataoka, Toru Nakayama, Masaki Yamamoto, Seiji Takahashi, Satoshi Yamashita

ChemBioChem　2024/01/29
Publisher: Wiley
DOI： 10.1002/cbic.202300796 　

ISSN： 1439-4227

eISSN： 1439-7633

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Abstract Neryl diphosphate (C₁₀) synthase (NDPS1), a homodimeric soluble cis‐prenyltransferase from tomato, contains four disulfide bonds, including two inter‐subunit S−S bonds in the N‐terminal region. Mutagenesis studies demonstrated that the S−S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C‐terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well‐ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C‐terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site‐directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N‐terminal regions and the closed/open states of C‐terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.
Structural insights into a bacterial β-glucosidase capable of degrading sesaminol triglucoside to produce sesaminol: Toward the understanding of the aglycone recognition mechanism by the C-terminal lid domain

Taro Yanai, Yukino Takahashi, Eri Katsumura, Naoki Sakai, Kohei Takeshita, Riki Imaizumi, Hiroaki Matsuura, Shuntaro Hongo, Toshiyuki Waki, Seiji Takahashi, Masaki Yamamoto, Kunishige Kataoka, Toru Nakayama, Satoshi Yamashita

The Journal of Biochemistry　174　(4)　335-344　2023/06/29
Publisher: Oxford University Press (OUP)
DOI： 10.1093/jb/mvad048 　

ISSN： 0021-924X

eISSN： 1756-2651

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Abstract The sesaminol triglucoside (STG)-hydrolyzing β-glucosidase from Paenibacillus sp. (PSTG1), which belongs to glycoside hydrolase family 3 (GH3), is a promising catalyst for the industrial production of sesaminol. We determined the X-ray crystal structure of PSTG1 with bound glycerol molecule in the putative active site. PSTG1 monomer contained typical three domains of GH3 with the active site in domain 1 (TIM barrel). In addition, PSTG1 contained an additional domain (domain 4) at the C-terminus that interacts with the active site of the other protomer as a lid in the dimer unit. Interestingly, the interface of domain 4 and the active site forms a hydrophobic cavity probably for recognizing the hydrophobic aglycone moiety of substrate. The short flexible loop region of TIM barrel was found to be approaching the interface of domain 4 and the active site. We found that n-heptyl-β-D-thioglucopyranoside detergent acts as an inhibitor for PSTG1. Thus, we propose that the recognition of hydrophobic aglycone moiety is important for PSTG1-catalyzed reactions. Domain 4 might be a potential target for elucidating the aglycone recognition mechanism of PSTG1 as well as for engineering PSTG1 to create a further excellent enzyme to degrade STG more efficiently to produce sesaminol.
A Polyphenol Oxidase Catalyzes Aurone Synthesis in Marchantia polymorpha

Hiraku Furudate, Misaki Manabe, Haruka Oshikiri, Ayako Matsushita, Bunta Watanabe, Toshiyuki Waki, Toru Nakayama, Hiroyoshi Kubo, Kojiro Takanashi

Plant and Cell Physiology　2023/03/22
Publisher: Oxford University Press (OUP)
DOI： 10.1093/pcp/pcad024 　

ISSN： 0032-0781

eISSN： 1471-9053

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Abstract Aurones constitute one of the major classes of flavonoids, with a characteristic furanone structure that acts as the C-ring of flavonoids. Members of various enzyme families are involved in aurone biosynthesis in different higher plants, suggesting that, during evolution, plants acquired the ability to biosynthesize aurones independently and convergently. Bryophytes also produce aurones, but the biosynthetic pathways and enzymes involved have not been determined. The present study describes the identification and characterization of a polyphenol oxidase that acts as an aureusidin synthase (MpAS1) in the model liverwort, Marchantia polymorpha. Crude enzyme assays using an M. polymorpha line overexpressing MpMYB14 with high accumulation of aureusidin showed that aureusidin was biosynthesized from naringenin chalcone and converted to riccionidin A. This activity was inhibited by N-phenylthiourea, an inhibitor specific to enzymes of the polyphenol oxidase family. Of the six polyphenol oxidases highly induced in the line overexpressing MpMyb14, one, MpAS1 was found to biosynthesize aureusidin from naringenin chalcone when expressed in Saccharomyces cerevisiae. MpAS1 also recognized eriodictyol chalcone, isoliquiritigenin and butein, showing the highest activity for eriodictyol chalcone. Members of the polyphenol oxidase family in M. polymorpha evolved independently from polyphenol oxidases in higher plants, indicating that aureusidin synthases evolved in parallel in land plants.
Structure‐based engineering of a short‐chain cis ‐prenyltransferase to biosynthesize nonnatural all‐ cis ‐polyisoprenoids: molecular mechanisms for primer substrate recognition and ultimate product chain‐length determination

Ryo Kutsukawa, Riki Imaizumi, Miki Suenaga‐Hiromori, Kohei Takeshita, Naoki Sakai, Shuto Misawa, Masaki Yamamoto, Haruhiko Yamaguchi, Yukino Miyagi‐Inoue, Toshiyuki Waki, Kunishige Kataoka, Toru Nakayama, Satoshi Yamashita, Seiji Takahashi

The FEBS Journal　2022/02/22
Publisher: Wiley
DOI： 10.1111/febs.16392 　

ISSN： 1742-464X

eISSN： 1742-4658
(+)-Sesamin, a sesame lignan, is a potent inhibitor of gut bacterial tryptophan indole-lyase that is a key enzyme in chronic kidney disease pathogenesis. International-journal

Daiki Oikawa, Satoshi Yamashita, Seiji Takahashi, Toshiyuki Waki, Koichi Kikuchi, Takaaki Abe, Takane Katayama, Toru Nakayama

Biochemical and biophysical research communications　590　158-162　2022/01/29

DOI： 10.1016/j.bbrc.2021.12.088 　

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The progression of chronic kidney disease (CKD) increases the risks of cardiovascular morbidity and end-stage kidney disease. Indoxyl sulfate (IS), which is derived from dietary l-tryptophan by the action of bacterial l-tryptophan indole-lyase (TIL) in the gut, serves as a uremic toxin that exacerbates CKD-related kidney disorder. A mouse model previously showed that inhibition of TIL by 2-aza-l-tyrosine effectively reduced the plasma IS level, causing the recovery of renal damage. In this study, we found that (+)-sesamin and related lignans, which occur abundantly in sesame seeds, inhibit intestinal bacteria TILs. Kinetic studies revealed that (+)-sesamin and sesamol competitively inhibited Escherichia coli TIL (EcTIL) with Ki values of 7 μM and 14 μM, respectively. These Ki values were smaller than that of 2-aza-l-tyrosine (143 μM). Molecular docking simulation of (+)-sesamin- (or sesamol-)binding to EcTIL predicted that these inhibitors potentially bind near the active site of EcTIL, where the cofactor pyridoxal 5'-phosphate is bound, consistent with the kinetic results. (+)-Sesamin is a phytochemical with a long history of consumption and is generally regarded as safe. Hence, dietary supplementation of (+)-sesamin encapsulated in enteric capsules could be a promising mechanism-based strategy to prevent CKD progression. Moreover, the present findings would provide a new structural basis for designing more potent TIL inhibitors for the development of mechanism-based therapeutic drugs to treat CKD.
Comprehensive identification of terpene synthase genes and organ-dependent accumulation of terpenoid volatiles in a traditional medicinal plant Angelica archangelica L.

Miki Suenaga-Hiromori, Daisuke Mogi, Yohei Kikuchi, Jiali Tong, Naotsugu Kurisu, Yuichi Aoki, Hiroyuki Amano, Masahiro Furutani, Takefumi Shimoyama, Toshiyuki Waki, Toru Nakayama, Seiji Takahashi

Plant Biotechnology　39　(4)　391-404　2022

DOI： 10.5511/plantbiotechnology.22.1006a 　

ISSN： 1342-4580

eISSN： 1347-6114
Identification of the Genes Coding for Carthamin Synthase, Peroxidase Homologs that Catalyze the Final Enzymatic Step of Red Pigmentation in Safflower (Carthamus tinctorius L.).

Toshiyuki Waki, Miho Terashita, Naoki Fujita, Keishi Fukuda, Mikiya Kato, Takashi Negishi, Hiromi Uchida, Yuichi Aoki, Seiji Takahashi, Toru Nakayama

Plant & cell physiology　2021/08/03

DOI： 10.1093/pcp/pcab122 　

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Carthamin, a dimeric quinochalcone that is sparingly soluble in water, is obtained from the yellow-orange corolla of fully blooming safflower (Carthamus tinctorius L.) florets. Carthamin is a natural red colorant, which has been used worldwide for more than 4500 years and is the major component of Japanese "beni" used for dyeing textiles, cosmetics, and as a food colorant. The biosynthetic pathway of carthamin has long remained uncertain. Previously, carthamin was proposed to be derived from precarthamin (PC), a water-soluble quinochalcone, via a single enzymatic process. In this study, we identified the genes coding for the enzyme responsible for formation of carthamin from PC, termed carthamin synthase (CarS), using enzyme purification and transcriptome analysis. The CarS proteins were purified from the cream-colored corolla of safflower and identified as peroxidase homologs (CtPOD1, CtPOD2, and CtPOD3). The purified enzyme catalyzed the oxidative decarboxylation of PC to produce carthamin using O2, instead of H2O2, as an electron acceptor. In addition, CarS catalyzed the decomposition of carthamin. However, this enzymatic decomposition of carthamin could be circumvented by adsorption of the pigment to cellulose. These CtPOD isozymes were not only expressed in the corolla of the carthamin-producing orange safflower cultivars, but were also abundantly expressed in tissues and organs that did not produce carthamin and PC. One CtPOD isozyme, CtPOD2, was localized in the extracellular space. Based on the results obtained, a model for the stable red pigmentation of safflower florets during flower senescence and the traditional "beni" manufacturing process is proposed.
Managing enzyme promiscuity in plant specialized metabolism: A lesson from flavonoid biosynthesis: Mission of a "body double" protein clarified. International-journal

Toshiyuki Waki, Seiji Takahashi, Toru Nakayama

BioEssays : news and reviews in molecular, cellular and developmental biology　43　(3)　e2000164　2021/03

DOI： 10.1002/bies.202000164 　

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Specificities of enzymes involved in plant specialized metabolism, including flavonoid biosynthesis, are generally promiscuous. This enzyme promiscuity has served as an evolutionary basis for new enzyme functions and metabolic pathways in land plants adapting to environmental challenges. This phenomenon may lead, however, to inefficiency in specialized metabolism and adversely affect metabolite-mediated plant survival. How plants manage enzyme promiscuity for efficient specialized metabolism is, thus, an open question. Recent studies of flavonoid biosynthesis addressing this issue have revealed a conserved strategy, namely, a homolog of chalcone isomerase with no catalytic activity binds to chalcone synthase, a key flavonoid pathway enzyme, to narrow (or rectify) the enzyme's highly promiscuous product specificity. Reducing promiscuity via specific protein-protein interactions among metabolic enzymes and proteins may be a solution adopted by land plants to achieve efficient operation of specialized metabolism, while the intrinsic promiscuity of enzymes has likely been retained incidentally.
Alteration of oxidative-stress and related marker levels in mouse colonic tissues and fecal microbiota structures with chronic ethanol administration: Implications for the pathogenesis of ethanol-related colorectal cancer. International-journal

Hideo Ohira, Atsuki Tsuruya, Daiki Oikawa, Wao Nakagawa, Rie Mamoto, Masahira Hattori, Toshiyuki Waki, Seiji Takahashi, Yoshio Fujioka, Toru Nakayama

PloS one　16　(2)　e0246580　2021

DOI： 10.1371/journal.pone.0246580 　

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Chronic ethanol consumption is a risk factor for colorectal cancer, and ethanol-induced reactive oxygen species have been suggested to play important roles in the pathogenesis of ethanol-related colorectal cancer (ER-CRC). In this study, the effects of 10-week chronic administration of ethanol on the colonic levels of oxidative stress and advance glycation end product (AGE) levels, as well as fecal microbiota structures, were examined in a mouse model. Chronic oral administration of ethanol in mice (1.0 mL of 1.5% or 5.0% ethanol (v/v) per day per mouse, up to 10 weeks) resulted in the elevation of colonic levels of oxidative stress markers (such as 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal) compared to control mice, and this was consistently accompanied by elevated levels of inflammation-associated cytokines and immune cells (Th17 and macrophages) and a decreased level of regulatory T (Treg) cells to produce colonic lesions. It also resulted in an alteration of mouse fecal microbiota structures, reminiscent of the alterations observed in human inflammatory bowel disease, and this appeared to be consistent with the proposed sustained generation of oxidative stress in the colonic environment during chronic ethanol consumption. Moreover, the first experimental evidence that chronic ethanol administration results in elevated levels of advanced glycation end products (AGEs) and their receptors (RAGE) in the colonic tissues in mice is also shown, implying enhanced RAGE-mediated signaling with chronic ethanol administration. The RAGE-mediated signaling pathway has thus far been implicated as a link between the accumulation of AGEs and the development of many types of chronic colitis and cancers. Thus, enhancement of this pathway likely exacerbates the ethanol-induced inflammatory states of colonic tissues and might at least partly contribute to the pathogenesis of ER-CRC.
Crystal structure of chalcone synthase, a key enzyme for isoflavonoid biosynthesis in soybean. International-journal Peer-reviewed

Riki Imaizumi, Ryo Mameda, Kohei Takeshita, Hiroki Kubo, Naoki Sakai, Shun Nakata, Seiji Takahashi, Kunishige Kataoka, Masaki Yamamoto, Toru Nakayama, Satoshi Yamashita, Toshiyuki Waki

Proteins　2020/07/29

DOI： 10.1002/prot.25988 　

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Isoflavonoid is one of the groups of flavonoids that play pivotal roles in the survival of land plants. Chalcone synthase (CHS), the first enzyme of the isoflavonoid biosynthetic pathway, catalyzes the formation of a common isoflavonoid precursor. We have previously reported that an isozyme of soybean CHS (termed GmCHS1) is a key component of the isoflavonoid metabolon, a protein complex to enhance efficiency of isoflavonoid production. Here, we determined the crystal structure of GmCHS1 as a first step of understanding the metabolon structure, as well as to better understand the catalytic mechanism of GmCHS1.
A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity. International-journal Peer-reviewed

Toshiyuki Waki, Ryo Mameda, Takuya Nakano, Sayumi Yamada, Miho Terashita, Keisuke Ito, Natsuki Tenma, Yanbing Li, Naoto Fujino, Kaichi Uno, Satoshi Yamashita, Yuichi Aoki, Konstantin Denessiouk, Yosuke Kawai, Satoko Sugawara, Kazuki Saito, Keiko Yonekura-Sakakibara, Yasumasa Morita, Atsushi Hoshino, Seiji Takahashi, Toru Nakayama

Nature communications　11　(1)　870-870　2020/02/13

DOI： 10.1038/s41467-020-14558-9 　

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Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.
Glycoside-specific glycosyltransferases catalyze regio-selective sequential glucosylations for a sesame lignan, sesaminol triglucoside. International-journal Peer-reviewed

Ono E, Waki T, Oikawa D, Murata J, Shiraishi A, Toyonaga H, Kato M, Ogata N, Takahashi S, Yamaguchi MA, Horikawa M, Nakayama T

The Plant journal : for cell and molecular biology　101　(5)　1221-1233　2019/10

DOI： 10.1111/tpj.14586 　

ISSN： 0960-7412

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Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid-related plant specialized metabolites. (+)-Sesamin and (+)-sesamolin are major hydrophobic lignans, whereas (+)-sesaminol primarily accumulates as a water-soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6-O-glucosidic linkages [i.e. (+)-sesaminol 2-O-β-d-glucosyl-(1→2)-O-β-d-glucoside-(1→6)-O-β-d-glucoside]. We previously reported that the 2-O-glucosylation of (+)-sesaminol aglycon and β1→6-O-glucosylation of (+)-sesaminol 2-O-β-d-glucoside (SMG) are mediated by UDP-sugar-dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2-O-glucosylation of SMG and (+)-sesaminol 2-O-β-d-glucosyl-(1→6)-O-β-d-glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside-specific glycosyltransferases (GGTs) and co-ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG-deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar-sugar β1→6-O-glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two-hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane-associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane-bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β-O-glucosylation sequence of STG biosynthesis is β1→2-O-glucosylation of SMG by UGT94AG1 followed by UGT94AA2-mediated β1→6-O-glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio-selectivity of sequential glucosylations catalyzed by GGTs.
Formation of Flavonoid Metabolons: Functional Significance of Protein-Protein Interactions and Impact on Flavonoid Chemodiversity Peer-reviewed

Toru Nakayama, Seiji Takahashi, Toshiyuki Waki

Frontiers in Plant Science　10　821　2019/07/09
Publisher: Frontiers Media SA
DOI： 10.3389/fpls.2019.00821 　

eISSN： 1664-462X
Identification and characterization of a novel bacterial β-glucosidase that is highly specific for the β-1,2-glucosidic linkage of sesaminol triglucoside. Peer-reviewed

Sakurai A, Hongo S, Nair A, Waki T, Oikawa D, Nishio T, Shimoyama T, Takahashi S, Yamashita S, Nakayama T

Bioscience, biotechnology, and biochemistry　82　(9)　1518-1521　2018/09

DOI： 10.1080/09168451.2018.1476123 　

ISSN： 0916-8451
Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2-hydroxyisoflavanone synthase. Peer-reviewed

Mameda R, Waki T, Kawai Y, Takahashi S, Nakayama T

The Plant journal : for cell and molecular biology　96　(1)　56-74　2018/07

DOI： 10.1111/tpj.14014 　

ISSN： 0960-7412
Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species Peer-reviewed

Naoto Fujino, Natsuki Tenma, Toshiyuki Waki, Keisuke Ito, Yuki Komatsuzaki, Keigo Sugiyama, Tatsuya Yamazaki, Saori Yoshida, Masayoshi Hatayama, Satoshi Yamashita, Yoshikazu Tanaka, Reiko Motohashi, Konstantin Denessiouk, Seiji Takahashi, Toru Nakayama

Plant Journal　94　(2)　372-392　2018/04/01
Publisher: Blackwell Publishing Ltd
DOI： 10.1111/tpj.13864 　

ISSN： 1365-313X 0960-7412
Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis Peer-reviewed

Satoshi Yamashita, Haruhiko Yamaguchi, Toshiyuki Waki, Yuichi Aoki, Makie Mizuno, Fumihiro Yanbe, Tomoki Ishii, Ayuta Funaki, Yuzuru Tozawa, Yukino Miyagi-Inoue, Kazuhisa Fushihara, Toru Nakayama, Seiji Takahashi

ELIFE　5　e19022　2016/10

DOI： 10.7554/eLife.19022 　

ISSN： 2050-084X
Transformation and isoflavonoid analyses of suspension-cultured cells of soybean [Glycine max (L.) Merr. cv. Enrei] Peer-reviewed

Toshiyuki Waki, Seiji Takahashi, Toru Nakayama

PLANT BIOTECHNOLOGY　33　(2)　137-141　2016/06

DOI： 10.5511/plantbiotechnology.16.0515a 　

ISSN： 1342-4580
Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.) Peer-reviewed

Toshiyuki Waki, DongChan Yoo, Naoto Fujino, Ryo Mameda, Konstantin Denessiouk, Satoshi Yamashita, Reiko Motohashi, Tomoyoshi Akashi, Toshio Aoki, Shin-ichi Ayabe, Seiji Takahashi, Toru Nakayama

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　469　(3)　546-551　2016/01

DOI： 10.1016/j.bbrc.2015.12.038 　

ISSN： 0006-291X

eISSN： 1090-2104
Identification of a Highly Specific Isoflavone 7-O-glucosyltransferase in the soybean (Glycine max (L.) Merr.) Peer-reviewed

Ayuta Funaki, Toshiyuki Waki, Akio Noguchi, Yosuke Kawai, Satoshi Yamashita, Seiji Takahashi, Toru Nakayama

PLANT AND CELL PHYSIOLOGY　56　(8)　1512-1520　2015/08

DOI： 10.1093/pcp/pcv072 　

ISSN： 0032-0781

eISSN： 1471-9053
Transcription analyses of GmICHG, a gene coding for a beta-glucosidase that catalyzes the specific hydrolysis of isoflavone conjugates in Glycine max (L.) Merr Peer-reviewed

DongChan Yoo, Takayuki Hara, Naoki Fujita, Toshiyuki Waki, Akio Noguchi, Seiji Takahashi, Toru Nakayama

PLANT SCIENCE　208　10-19　2013/07

DOI： 10.1016/j.plantsci.2013.03.006 　

ISSN： 0168-9452

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Misc. 53

The Crystal Structure of Chalcone Synthase Complexd with Chalcone Isomerase-like Protein

今泉璃城, 和氣駿之, 竹下浩平, 安田あおい, 松浦滉明, 中多舜, 坂井直樹, 片岡邦重, 高橋征司, 山本雅貴, 山下哲, 中山亨

日本農芸化学会大会講演要旨集(Web)　2024　2024

ISSN： 2186-7976
多酵素複合体(メタボロン)によるフラボノイド代謝調節機構

和氣駿之

日本生薬学会年会講演要旨集　70th　2024

ISSN： 0919-1992
ダイズおよびキンギョソウにおけるアントシアニジン合成酵素の機能解析

和氣駿之, 垣生大希, 斎藤泰知, 古川楓, 高橋征司, 吉田久美, 中山亨

日本農芸化学会東北支部大会プログラム・講演要旨集　159th (Web)　2024
The Crystal Structure of Sesame Lignan Glycoside Hydrolase and the Novel Function of its C-terminal Domain

矢内太朗, 高橋由季乃, 坂井直樹, 竹下浩平, 今泉璃城, 松浦滉明, 和氣駿之, 高橋征司, 山本雅貴, 片岡邦重, 中山亨, 山下哲

日本農芸化学会大会講演要旨集(Web)　2024　2024

ISSN： 2186-7976
フラボノイド生合成を制御するメタボロン

和氣駿之

生物工学会誌　101　(9)　493-496　2023/09

DOI： 10.34565/seibutsukogaku.101.9_493 　
比較機能解析によるセイヨウトウキ由来テルペン合成酵素の生成物特異性決定残基の探索

天野博之, 栗栖尚嗣, 角掛陽, 茂木大介, 菊池洋平, 廣森美樹, 和氣駿之, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集(CD-ROM)　33rd　2023
サポジラ(Manilkara zapota)由来トランス型プレニルトランスフェラーゼのタンパク質工学とX線結晶構造解析の試み

矢内太朗, 森麻人, 中切亮我, 今泉璃城, 山口晴彦, 竹下浩平, 松浦滉明, 宮城ゆき乃, 片岡邦重, 和氣駿之, 中山亨, 高橋征司, 山下哲

イソプレノイド研究会例会講演要旨集(CD-ROM)　33rd　2023
セサミノール配糖体加水分解酵素のX線結晶構造解析によって示されたC末端ドメインの新奇機能

矢内太朗, 高橋由季乃, 坂井直樹, 竹下浩平, 今泉璃城, 松浦滉明, 宮原一真, 和氣駿之, 高橋征司, 山本雅貴, 片岡邦重, 中山亨, 山下哲

日本生化学会大会(Web)　96th　2023
Identification and characterization of C-glycosyltransferases from Carthamus tinctorius L.

門脇芽以, 和氣駿之, 藤田直樹, 沼野井一輝, 佐藤誠哉, 寺下美穂, 福田敬志, 加藤幹也, 根岸尚志, 内田弘美, 青木裕一, 田口悟朗, 高橋征司, 中山亨

日本植物生理学会年会(Web)　64th　2023
Solubilization of natural rubber biosynthetic enzyme complexes from rubber particles of the Para rubber tree (Hevea brasiliensis) by the amphiphilic copolymer treatments

KHAN Nadia Nur Shazana Binti Abu Talib, 小島幸治, 山口晴彦, 三上智世, 廣森美樹, 和氣駿之, 宮城ゆき乃, 山下哲, 戸澤譲, 中山亨, 高橋征司

日本植物生理学会年会(Web)　64th　2023
フラボノイド生合成における縁の下の力持ち

和氣駿之

生物工学会誌　100　(5)　254-254　2022/05/25
Publisher: 公益社団法人日本生物工学会
DOI： 10.34565/seibutsukogaku.100.5_254 　

ISSN： 0919-3758

eISSN： 2435-8630
高等植物のシス型プレニルトランスフェラーゼの活性制御におけるcPT-like proteinの役割

KHAN Nadia Nur Shazana Binti Abu Talib, 高橋朋宏, 廣森美樹, 山口真琴, 皆川知歩, 山家史大, 大場崇史, 山下哲, 戸澤譲, 山口晴彦, 宮城ゆき乃, 和氣駿之, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　32nd (CD-ROM)　2022
黒ダイズ(Glycine max)のアントシアニン生合成関連酵素群の機能解析

斎藤泰知, 古川楓, 澤口玲央, 和氣駿之, 吉田久美, 中山亨

日本農芸化学会東北支部大会プログラム・講演要旨集　2022　2022
脂肪滴およびゴム粒子の構造維持に関わる膜タンパク質の比較解析

中山隆司, 開琢海, 三輪幸祐, 廣森美樹, 和氣駿之, 山下哲, 戸澤譲, 山口晴彦, 宮城ゆき乃, 岩井雅子, 太田啓之, 中山亨, 高橋征司

日本生化学会大会(Web)　95th　2022
セサミノール配糖体加水分解酵素の推定触媒残基の機能解析

高橋由季乃, 矢内太朗, 勝村恵理, 和氣駿之, 片岡邦重, 中山亨, 山下哲

日本生化学会大会(Web)　95th　2022
フラボノイド生合成の初発酵素であるカルコン合成酵素の阻害に関する構造的研究

安田あおい, 今泉璃城, 中多舜, 竹下浩平, 坂井直樹, 和氣駿之, 片岡邦重, 中山亨, 山下哲

日本生化学会大会(Web)　95th　2022
Elucidation of molecular mechanisms for product specificity of terpene synthases by comparative functional analyses

天野博之, 栗栖尚嗣, 山家史大, 廣森美樹, 和氣駿之, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2022　2022

ISSN： 2186-7976
Identification and functional analysis of interaction domains of rubber transferase protein complex from Hevea brasiliensis

NUR SHAZANA BINTI ABU TALIB KHAN Nadia, 廣森美樹, 和氣駿之, 山下哲, 戸澤譲, 山口晴彦, 宮城ゆき乃, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2022　2022

ISSN： 2186-7976
Phosphorylation-mediated regulation of eukaryotic cis-prenyltransferases contributing to dolichol biosynthesis

高橋朋宏, 山家史大, 酒井勇貴, 皆川知歩, 和氣駿之, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2022　2022

ISSN： 2186-7976
A new biosynthetic pathway of cyanidin 3-O-glucoside in seed coat of black soybean, Glycine max

澤口玲央, 古川楓, 和氣駿之, TEPPABUT Yada, 中根悠輔, 林英美, 尾山公一, 杉田千恵子, 近藤忠雄, 中山亨, 吉田久美

天然有機化合物討論会講演要旨集(Web)　63rd　2021

ISSN： 2433-1856
Elucidation of catalytic mechanisms for monoterpene synthases by comparative functional analyses

栗栖尚嗣, 角掛陽, 茂木大介, 菊池洋平, 廣森美樹, 和氣駿之, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2021　2021

ISSN： 2186-7976
ドリコール生合成に寄与するcis型プレニルトランスフェラーゼの翻訳後修飾による活性制御

高橋朋宏, 山家史大, 酒井勇貴, 皆川知歩, 和氣駿之, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　31st (CD-ROM)　2021
Establishment of a monoterpenoid overproduction system by pathway engineering of Escherichia coli

宮下孝洋, 宮下孝洋, 栗栖尚嗣, 角掛陽, 和氣駿之, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2021　2021

ISSN： 2186-7976
ゴマ種子におけるセサミノール配糖体の代謝生理学

青沼聡, 工藤紫苑, 和氣駿之, 高橋征司, 中山亨

日本農芸化学会東北支部大会プログラム・講演要旨集　156th (CD-ROM)　2021
Metabolon formation in plant specialized metabolism

Nakayama Toru, Waki Toshiyuki, Takahashi Seiji

Regulation of Plant Growth & Development　56　(1)　14-25　2021
Publisher: The Japanese Society for Chemical Regulation of Plants
DOI： 10.18978/jscrp.56.1_14 　

ISSN： 1346-5406

eISSN： 2189-6305

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Plant produces a diverse array of metabolites, such as flavonoids, isoprenoids, and alkaloids. It has been shown that these metabolites play important roles in plant survival and reproduction in plat lineage-specific manners, with their structures differing with plant lineages. Thus, these metabolites are now called “plant specialized metabolites”. Biosynthetic pathways of many of these metabolites have been clarified, and it has been revealed that enzymes and proteins involved in their biosynthesis are found to form metabolons, which are complexes of weakly associated metabolic enzymes. This review surveys recent progress of metabolon research in plant specialized metabolism.
Exploring catalytically omportant amino acid residues of a soybean isoflavone glucosyltransferase

中山亨, 佐藤誠哉, 伊藤圭介, 高橋征司, 和氣駿之, 高橋厚人, 大山拓次, 楠木正巳, 今泉璃城, 山下哲

日本農芸化学会大会講演要旨集(Web)　2021　2021

ISSN： 2186-7976
In vitro biosynthesis of isoprenoid polymers by trans-prenyltransferases identified from Manilkara zapota

三輪幸祐, 廣森美樹, 青木裕一, 和氣駿之, 小島幸治, 山下哲, 山口晴彦, 宮城ゆき乃, 戸澤譲, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2021　2021

ISSN： 2186-7976
Establishment of a system for in vitro translation-coupled external protein introduction into lipid droplets from Chlamydomonas reinhardtii towards elucidation of lipid droplet transit sequence motifs.

開琢海, 廣森美樹, 和氣駿之, 山下哲, 戸澤譲, 山口晴彦, 宮城ゆき乃, 岩井雅子, 太田啓之, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2021　2021

ISSN： 2186-7976
Inhibition analysis and co-crystallization trial of sesaminol glycoside hydrolase with thioglucoside detergent

矢内太朗, 勝村恵理, 本江俊太郎, 和氣駿之, 片岡邦重, 高橋征司, 山下哲, 中山享

日本農芸化学会大会講演要旨集(Web)　2021　2021

ISSN： 2186-7976
サポジラ(Manilkara zapota)由来trans型プレニルトランスフェラーゼの酵素機能解析

三輪幸祐, 廣森美樹, 青木裕一, 和氣駿之, 小島幸治, 山下哲, 山口晴彦, 宮城ゆき乃, 戸澤譲, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　31st (CD-ROM)　2021
無細胞翻訳系を用いた脂肪滴への外来酵素導入によるイソプレノイドポリマー生産

開琢海, 三輪幸祐, 廣森美樹, 和氣駿之, 山下哲, 戸澤譲, 山口晴彦, 宮城ゆき乃, 岩井雅子, 太田啓之, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　31st (CD-ROM)　2021
フラボノイド生合成に進化的に保存されたカルコン合成酵素の活性制御機構とフラボノイドメタボロン（若手シンポジウム「ビタミン・バイオファクター研究の新潮流」）

和氣駿之

ビタミン　94　(4)　233　2020/04/25
Publisher: 公益社団法人日本ビタミン学会
DOI： 10.20632/vso.94.4_233 　

ISSN： 0006-386X

eISSN： 2424-080X
ダイズイソフラボノイド生合成に関与するカルコン合成酵素の結晶構造解析

中多舜, 和氣駿之, 今泉璃城, 久保大樹, 竹下浩平, 片岡邦重, 中山亨, 山下哲

日本生化学会大会(Web)　93rd　2020
X-ray crystallography of soybean isoflavone biosynthetic enzyme

山下哲, 今泉璃城, 中多舜, 竹下浩平, 和氣駿之, 山本雅貴, 高橋征司, 片岡邦重, 中山亨

日本農芸化学会大会講演要旨集(Web)　2020　2020

ISSN： 2186-7976
タンパク質間相互作用によるフラボノイド生合成酵素の活性制御:カルコン合成酵素の特異性あいまいさの矯正

和氣駿之, 高橋征司, 中山亨

酵素工学ニュ-ス　(84)　2020

ISSN： 0911-9957
Promiscuity of Enzyme Specificity and Evolution of Plant Specialized Metabolism: Implications from Flavonoid Biosynthesis: A Mission of a “Body Double” Protein

中山亨, 高橋征司, 和氣駿之

化学と生物　58　(6)　354-361　2020
Publisher: 日本農芸化学会 ; 1962-
ISSN： 0453-073X
ダイズの5‐デオキシ型イソフラボノイド生合成におけるメタボロン形成の役割細胞内フラボノイド代謝の高効率化

和氣駿之, 高橋征司, 中山亨

化学と生物　57　(10)　593‐595-595　2019/10/01
Publisher: Japan Society for Bioscience, Biotechnology, and Agrochemistry
DOI： 10.1271/kagakutoseibutsu.57.593 　

ISSN： 0453-073X

eISSN： 1883-6852
パラゴムノキの天然ゴム生合成マシナリを構成する因子の相互作用と機能相関

小島幸治, 山口真琴, 石井智樹, 廣森美樹, 和氣駿之, 山下哲, 戸澤譲, 山口春彦, 井之上ゆき乃, 伏原和久, 中山亨, 高橋征司

日本植物生理学会年会(Web)　60th　435 (WEB ONLY)　2019
パラゴムノキの天然ゴム合成反応におけるゴム粒子の役割

小島幸治, 廣森美樹, 山家史大, 石井智樹, 和氣駿之, 山下哲, 戸澤譲, 山口晴彦, 井之上ゆき乃, 伏原和久, 中山亨, 高橋征司

日本農芸化学会東北支部大会プログラム・講演要旨集　153rd　51　2018/09/22
パラゴムノキの天然ゴム生合成に関与するHRT1‐REF BRIDGING PROTEINの機能解析

山口真琴, 和氣駿之, 青木裕一, 山家史大, 石井智樹, 山下哲, 高橋征司, 中山亨

日本植物細胞分子生物学会大会・シンポジウム講演要旨集　36th　119　2018/08/15
天然ゴム生合成機構におけるゴム粒子の重要性

石井智樹, 末永美樹, 山下哲, 山家史大, 和氣駿之, 小島幸治, 山口晴彦, 井之上ゆき乃, 伏原和久, 中山亨, 高橋征司

日本農芸化学会大会講演要旨集(Web)　2018　ROMBUNNO.2A25a10 (WEB ONLY)　2018/03/05

ISSN： 2186-7976
The enzymatic study of hydroxylation, methylation and glycosylation of Phytolacca americana

濱田博喜, 上杉大介, 藤高侑也, 下田恵, 小崎紳一, 和氣駿之, 中山亨, 福田庸太, 井上豪

天然有機化合物討論会講演要旨集(Web)　60th　2018

ISSN： 2433-1856
Flavonoid metabolons

WAKI Toshiyuki, Seiji takahashi, Toru nakayama

BIOSCIENCE & INDUSTRY　76　(5)　390-394　2018
Publisher: バイオインダストリー協会
ISSN： 0914-8981
ゴム粒子上での天然ゴム合成について

山口晴彦, 宮城ゆき乃, 伏原和久, 山下哲, 青木裕一, 和氣駿之, 水野槙恵, 山家史大, 中山亨, 高橋征司

エラストマー討論会講演要旨集　28th　191‐192　2017/11/29
シス型プレニルトランスフェラーゼによる試験管内天然ゴム合成

山下哲, 石井智樹, 山家史大, 和氣駿之, 山口晴彦, 宮城ゆき乃, 伏原和久, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　27th　15　2017/09/15
高等植物のシス型プレニルトランスフェラーゼの活性制御におけるNgBRファミリータンパク質の役割

皆川知歩, 和氣駿之, 山家史大, 石井智樹, 解良康太, 平間匠, 工藤雅史, 山下哲, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　27th　17　2017/09/15
パラゴムノキのシス型プレニル鎖延長酵素ファミリーの包括的機能解析

石井智樹, 山家史大, 和氣駿之, 青木裕一, 山下哲, 高橋征司, 中山亨

日本植物細胞分子生物学会大会・シンポジウム講演要旨集　35th　87　2017/08/20
バイオ工学を駆使した天然ゴムの合成 : 生合成機構の解明と安定供給に大きく前進

和氣駿之, 山下哲, 中山亨, 高橋征司

化学 = Chemistry　72　(8)　12-16　2017/08
Publisher: 化学同人
ISSN： 0451-1964
セサミノール配糖体の新規分解酵素の単離およびキャラクタリゼーション

櫻井明徳, 山下哲, 山下哲, 和氣駿之, 高橋征司, 中山亨

日本生化学会大会(Web)　90th　ROMBUNNO.2LBA‐007 (WEB ONLY)　2017
バイオ資源の探索研究

中山亨, 和氣駿之, 高橋征司

CHEMISTRY & EDUCATION　65　(1)　12-15　2017
Publisher: 株式会社東京化学同人
DOI： 10.20665/kakyoshi.65.1_12 　

ISSN： 0386-2151
パラゴムノキの乳管特異的な遺伝子発現制御に寄与するタンパク質の探索

青木裕一, 船木亜由太, 和氣駿之, 山下哲, 高橋征司, 中山亨

イソプレノイド研究会例会講演要旨集　26th　23　2016/09/20
パラゴムノキの天然ゴム合成酵素の分子解析

山下哲, 山下哲, 山口晴彦, 和氣駿之, 山家史大, 青木裕一, 宮城ゆき乃, 伏原和久, 戸澤譲, 中山亨, 高橋征司

イソプレノイド研究会例会講演要旨集　26th　22　2016/09/20
ダイズのUGT88ファミリーグルコシルトランスフェラーゼの特異性の進化

船木亜由太, 和氣駿之, 山下哲, 桑名美貴子, 大山拓次, 楠木正巳, 高橋征司, 中山亨

日本植物細胞分子生物学会大会・シンポジウム講演要旨集　32nd　73　2014/07/30

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Presentations 12

A regulatory mechanism of flavonoid biosynthetic enzyme activity via protein-protein interaction Invited

Toshiyuki Waki

2023/09/13
Inhibition mechanism of chalcone synthase by CoA-SH

Toshiyuki Waki, Yamato doi, Kaichi Uno, Sayumi Yamada, Riki Imaizumi, Seiji Takahashi, Satoshi Yamashita, Toru Nakayama

2022/09/13
Identification and functional characterization of enzyme involved in carthamin biosynthesis in safflower (Carthamus tinctorius L.)

2021/09/10
フラボノイド生合成に進化的に保存されたカルコン合成酵素の活性制御機構とフラボノイドメタボロン

和氣駿之

日本ビタミン学会第72回大会　2020/09/04
Analysis of molecular mechanism involved in activation of chalcone synthase by means of Enhancer of Flavonoid Production

WAKI Toshiyuki

2019/03/26
Enhancer of Flavonoid Productionによるカルコン合成酵素の生成物特異性制御

和氣駿之

日本農芸化学会東北・北海道合同支部大会　2018/09/23
フラボノイド生合成における多酵素複合体（メタボロン）に関する研究

和氣駿之

日本農芸化学会東北・北海道合同若手の会　2018/09/21
Catalytic consequence of protein-protein interactions of chalcone synthase with enhancer of flavonoid production

WAKI Toshiyuki

2018/08/27
Enzymological characterizations of chalcone reductase paralogs of soybean

平成29年度化学系学協会東北大会　2017/09/16
ダイズ細胞内におけるイソフラボン代謝ダイナミクス

第10回ダイズ研究会　2017/03/10
フラボノイド生合成におけるメタボロン形成

第5回植物二次代謝フロンティア研究会　2016/11/05
ダイズにおけるイソフラボン代謝関連酵素の発現挙動，相互作用，アイソザイムの機能分化に関する研究

第34回日本植物細胞分子生物学会（上田）大会　2016/09/01

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Industrial Property Rights 2

EFPタンパク質を発現する大腸菌およびそれを用いたフラボノイド化合物製造方法

藤田直樹, 中山亨, 高橋征司, 和氣駿之

特許第6635535号

Property Type: Patent
新規カルコン色素およびその製造用の組成物と方法

藤田直樹, 中山亨, 高橋征司, 和氣駿之, 山田彩友美

Property Type: Patent

Research Projects 6

特化メタボロン・総括班

森貴裕, 佐々木栄太, 和氣駿之, 寺坂尚紘

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 学術変革領域研究(B)

Institution: 東京大学

2025/04/01 - 2028/03/31
Specialized metabolons confer structural diversity to plant specialized metabolites

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Transformative Research Areas (B)

Institution: Tohoku University

2025/04/01 - 2028/03/31
Molecular dissection of plant specialized metabolism machineries

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Scientific Research (S)

Institution: Tohoku University

2023/04/12 - 2028/03/31
ダイズ細胞内のイソフラボン配糖体の分解代謝機構の解明

和氣駿之

Offer Organization: 日本学術振興会

System: 科学研究費助成事業若手研究

Category: 若手研究

Institution: 東北大学

2022/04/01 - 2025/03/31
フラボノイド生合成の細胞内動態を探る

和氣駿之

Offer Organization: 日本科学技術振興機構

System: 戦略的創造研究推進事業ACT-X

Institution: 東北大学

2021/10 - 2024/03

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細胞内で生じる代謝は、連続する代謝酵素の複合体(メタボロン)の形成により時空間的に制御されていることが考えられています。本研究では、フラボノイド代謝酵素群に着目してメタボロン形成動態の解析を行い、タンパク質間相互作用を介した代謝酵素の特異性制御機構および基質チャネリングによる代謝効率化メカニズムを明らかにし、より環境負荷の小さい物質生産への応用を目指します。
陸上植物に進化的に保存されたカルコン合成酵素の活性制御機構の解明

和氣駿之

Offer Organization: 日本学術振興会

System: 科学研究費助成事業若手研究

Category: 若手研究

Institution: 東北大学

2020/04/01 - 2022/03/31

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カルコン合成酵素（CHS）とその相互作用タンパク質であるカルコン異性化酵素類似タンパク質（CHIL）の相互作用界面を明らかにするため，クロスリンカー試薬を用いてCHSとCHILを架橋し，架橋されたペプチドをLC-MSMSにより同定することでCHSとCHILの相互作用界面の同定を試みた．始めにクロスリンカー試薬の種類やタンパク質濃度，反応時間，用いるCHSおよびCHILの由来植物種の検討を行い，CHSとCHILが効果的に架橋される条件を探索した．SDS-PAGEにより架橋タンパク質を解析したところ，非常に低収率ながらCHSとCHILが1対1で架橋されたと推定されるタンパク質バンドを検出することができた．このタンパク質バンドをゲルから切り出した後，トリプシン処理を行いLC-MSMSにより架橋ペプチドを解析した．その結果，1対の架橋ペプチドが同定され，CHSとCHILの相互作用界面が推定できた．また，CHILによるCHSのカルコン生成の活性化メカニズムを調べるため，CHSとCoAの結晶構造を参照して，CoA結合に重要なアミノ酸残基にアラニンの変異を導入した変異型CHSを作製し，詳細な酵素機能解析を行った．3つのアミノ酸残基に着目してそれぞれの変異体を解析したところ，CoA感受性が増強された変異体，CHILによる活性矯正を受けなくなった変異体を取得することができた．各変異体の速度論解析から，CoA感受性が増強された変異体ではkcat値が大きく低下し，CHILの効果を受けなくなった変異体はkcat値およびKm値がともに大きく増強されたことが示された．これら結果から，CoA結合に関わるアミノ酸残基はCHSの酵素活性に大きく影響することが示された．

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