Faculty Profiles - TAMURA Takashi

MISC - TAMURA Takashi

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核酸系抗生物質シネフンギンの動物ウイルスに対する抗ウイルス活性評価

黒田雄大, 根本理子, 稲垣賢二, 田村隆, 前田健, 前田健

日本獣医学会学術集会講演要旨集 162nd 2019

[NiFeSe]型ヒドロゲナーゼのOne-Step精製を目的としたゲノム改変

小沼瞳, 山神将大, 袴塚響, 田嶋智之, 高口豊, 根本理子, 稲垣賢二, 田村隆

日本農芸化学会西日本支部大会およびシンポジウム講演要旨集 2019 2019

放線菌Streptomyces sp.590由来の抗腫瘍性酵素L‐メチオニン脱炭酸酵素の遺伝子クローニング,組換え発現,均一精製および性質検討

林将也, 岡田茜, 山本久美子, 奥河内知美, 日下知香, 工藤大蔵, 根本理子, 稲垣純子, 広瀬侑, 岡島俊英, 田村隆, 左右田健次, 稲垣賢二

ビタミン 92 ( 3 ) 113‐116 2018.3

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine gamma-lyase based on substrate-assisted catalysis

Dan Sato, Tomoo Shiba, Shunsuke Yunoto, Kazuo Furutani, Mitsuki Fukumoto, Daizou Kudou, Takashi Tamura, Kenji Inagaki, Shigeharu Harada

PROTEIN SCIENCE 26 ( 6 ) 1224 - 1230 2017.6

Language：English Publisher：WILEY

Methionine gamma-lyse (MGL) catalyzes the alpha, gamma-elimination of L-methionine and its derivatives as well as the alpha, beta-elimination of L-cysteine and its derivatives to produce alpha-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate-assisted catalysis.

DOI： 10.1002/pro.3158

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Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590. International journal

Masaya Hayashi, Akane Okada, Kumiko Yamamoto, Tomomi Okugochi, Chika Kusaka, Daizou Kudou, Michiko Nemoto, Junko Inagaki, Yuu Hirose, Toshihide Okajima, Takashi Tamura, Kenji Soda, Kenji Inagaki

Journal of biochemistry 161 ( 4 ) 389 - 398 2017.4

Language：English

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

DOI： 10.1093/jb/mvw083

PubMed

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L-メチオニン、L-ホモシステイン新規定量法へのL-メチオニン脱炭酸酵素の活用

林将也, 大川敦司, 半田暖尚, 根本理子, 稲垣純子, 菊池可菜子, 木村隆, 田村隆, 稲垣賢二

ビタミン 91 ( 4 ) 286 - 286 2017.4

The hyperthermophilic cystathionine gamma-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights

Dan Sato, Tomoo Shiba, Sae Mizuno, Ayaka Kawamura, Shoko Hanada, Tetsuya Yamada, Mai Shinozaki, Masahiko Yanagitani, Takashi Tamura, Kenji Inagaki, Shigeharu Harada

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 73 152 - 158 2017.3

Language：English Publisher：INT UNION CRYSTALLOGRAPHY

Cystathionine gamma-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P2(1); unit-cell parameters a = 58.4, b = 149.3, c = 90.2 angstrom , beta = 108.9 degrees) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C222(1); unit-cell parameters a = 117.7, b = 117.8, c = 251.3 angstrom ) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 angstrom ) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 angstrom resolution for forms I, II and III', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

DOI： 10.1107/S2053230X17002011

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海洋性細菌由来の新規キノン含有グリシンオキシダーゼ −組換え発現と性質検討−

溝端佐津紀, 根本理子, 田村隆, 稲垣賢二

2016年度ビタミンB研究委員会報告書 147 - 148 2017.2

Mechanism of PLP-dependent Aminomutases Elucidated by DFT-Calculation

91 ( 5-6 ) 355 - 360 2017

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp NT3060

Yasuhisa Fukuda, Asuka Abe, Takashi Tamura, Takahide Kishimoto, Atsushi Sogabe, Satoshi Akanuma, Shin-ichi Yokobori, Akihiko Yamagishi, Katsumi Imada, Kenji Inagaki

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 121 ( 5 ) 497 - 502 2016.5

Language：English Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (T-m), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60 degrees C (t(1/2)). Three mutants showed increased stabilities compared to the wild type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.09.011

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Identification and characterization of the zosA gene involved in copper uptake in Bacillus subtilis 168

Takahiro Fukuhara, Kazuo Kobayashi, Yousuke Kanayama, Shu-ichi Enomoto, Taeko Kondo, Naoki Tsunekawa, Michiko Nemoto, Naotake Ogasawara, Kenji Inagaki, Takashi Tamura

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 ( 3 ) 600 - 609 2016.3

Language：English Publisher：TAYLOR & FRANCIS LTD

DL-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu2+ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu2+ but not of excessive Zn2+ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.

DOI： 10.1080/09168451.2015.1107462

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1.放線菌由来の低基質特異性L-アミノ酸オキシダーゼの性質と構造(第442回研究協議会研究発表要旨,ビタミンB研究委員会)

村上佳穂, 伊藤菜々子, 今田勝巳, 山田美和, 礒部公安, 田村隆, 稲垣賢二

ビタミン 90 ( 1 ) 39 - 40 2016.1

海洋性細菌が産生する新規なキノン含有アミノ酸酸化酵素の利用に向けた研究

稲垣賢二, 赤地周作, 溝端佐津紀, 田村隆

低炭素社会と食の安全・安心を統合した環境生命科学的研究成果報告書 23 - 25 2016

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8

Saho Kamada, Takahiro Okugochi, Kaori Asano, Ryuta Tobe, Hisaaki Mihara, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 ( 10 ) 1970 - 1972 2016

Language：English Publisher：TAYLOR & FRANCIS LTD

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-C-14] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K-M and k(cat) for Sephs2-Sec60Cys were determined to be 26M and 0.352min(-1), respectively.

DOI： 10.1080/09168451.2016.1200458

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大学生の朝食欠食習慣の統計解析と改善への新指針

田村,隆, 揖斐, 隆之, 稲垣,賢二, 久保,康隆, 奥田,潔

岡山大学農学部学術報告 105 1 - 5 2016

放線菌由来の低基質特異性L-アミノ酸オキシダーゼの性質と構造

村上佳穂, 伊藤菜々子, 今田勝巳, 山田美和, 磯部公安, 田村隆, 稲垣賢二

2015年度ビタミンB研究委員会報告書 22 - 23 2016

Quantum Chemistry in Elucidation of the Reaction Mechanisms of PLP-dependent Enzymes

89 ( 5 ) 281 - 285 2016

Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride

Amano Marie, Mizuguchi Haruka, Sano Tadahisa, Kondo Hiroki, Shinyashiki Kengo, Inagaki Junko, Tamura Takashi, Kawaguchi Tatsuya, Kusakabe Hitoshi, Imada Katsumi, Inagaki Kenji

Vitamins 89 ( 10 ) 495 - 498 2015.10

Rhodococcus sp.AIU Z‐35‐1由来L‐アミノ酸酸化酵素―組換え酵素の精製及び性質―

村上佳穂, 橋本義輝, 小林達彦, 山田美和, 磯部公安, 根本理子, 田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 67th 275 - 275 2015.9

2-IV-24 海洋性細菌Marinomonas mediterranea由来グリシンオキシダーゼの大腸菌発現系の構築(一般演題要旨,ビタミン・バイオファクター研究のさらなる魅力〜大和まほろぱからの発信〜,第67回大会講演要旨)

溝端佐津紀, 赤地周作, 田村隆, 稲垣賢二

ビタミン 89 ( 4 ) 263 - 263 2015.4

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