研究者詳細 - 田村　隆

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年4月

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年3月

記述言語：英語出版者・発行元：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

ラジカル転移反応を触媒するピリドキサール酵素の量子化学計算

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年2月

田村隆

ビタミン 91 ( 5-6 ) 355 - 360 2017年

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年5月

記述言語：英語出版者・発行元：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

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年3月

記述言語：英語出版者・発行元：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

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年1月

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年

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年

記述言語：英語出版者・発行元：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

量子化学計算が解き明かすピリドキサール酵素の反応機構

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年

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年

田村隆

ビタミン 89 ( 5 ) 281 - 285 2016年

糸状菌Trichoderma viride由来の抗腫瘍性酵素L-リシン α-オキシダーゼの組換え発現,分子特性及び結晶構造の解析(研究論文紹介)

天野万里, 水口春香, 佐野宰久, 近藤裕輝, 新屋敷健吾, 稲垣純子, 田村隆, 川口辰也, 日下部均, 今田勝巳, 稲垣賢二

ビタミン 89 ( 10 ) 495 - 498 2015年10月

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年9月

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年4月

2-IV-20 組換えL-メチオニン脱炭酸酵素の精製および性質検討(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)

林将也, 岡田茜, 田村隆, 稲垣賢二

ビタミン 89 ( 4 ) 261 - 261 2015年4月

放線菌Streptomyces incarnatus NRRL8089のドラフトゲノムシーケンス

清水瞳, 大島健志朗, 服部正平, 稲垣賢二, 田村隆

日本農芸化学会大会講演要旨集(Web) 2015 3A37P07 (WEB ONLY) 2015年3月

Rhodococcus属放線菌由来低基質特異性L‐アミノ酸酸化酵素の遺伝子クローニングと発現系の構築

村上佳穂, 田港静, 橋本義輝, 小林達彦, 山田美和, 礒部公安, 田村隆, 稲垣賢二

日本農芸化学会大会講演要旨集(Web) 2015 2C22P14 (WEB ONLY) 2015年3月

海洋細菌Marinomonas mediterranea由来L‐リシンε‐オキシダーゼの大腸菌発現系の構築

赤地周作, 松井大亮, 浅野泰久, 田村隆, 稲垣賢二

日本農芸化学会大会講演要旨集(Web) 2015 2C22P15 (WEB ONLY) 2015年3月

部位特異的変異によるピラノース酸化酵素の色素依存性脱水素酵素活性の向上

荒木俊雄, 中柄朋子, 田村隆, 稲垣賢二

岡山大学農学部学術報告 104 1 - 4 2015年2月

記述言語：日本語出版者・発行元：岡山大学農学部

Pyranose oxidase (EC 1.1.3.10 ; PROD) catalyzes the oxidation of aldopyranoses at the position C‒2to yield the corresponding 2‒keto-aldoses and H2O2 , using oxygen as an electron acceptor. The enzyme shows broad substrate specificity as well as reactivity for 1,5‒anhydro‒d‒glucitol (1,5‒AG), which is known as a clinical glycemic marker. It is considered that the reactivity of PROD for 1,5‒AG is useful in the development of an amperometric-type biosensor, which is a convenient diagnostic device for selfmonitoringblood glucose (SMBG). However, the levels of dissolved oxygen in blood affect biosensorsystems that are equipped with an artificial electron mediator. In the present study, we attempted to develop an O2‒insensitive oxidase that would improve the dye-mediated dehydrogenase activity. We performed site-directed mutagenesis on PROD isolated from basidiomycetous fungus No. 52, which generated 11 mutants. The amino acid substitution Q421A exhibited a significant decrease (8.8% of wild type) in its oxidase activity, whereas it maintained its dehydrogenase activity (67% of wild type). In this study, we characterized PROD mutants from basidiomycetous fungus No. 52, which showed improved dye-mediated dehydrogenase activity.

抗腫瘍性L-リシンα-オキシダーゼの異種発現系構築と基質複合体のX線結晶構造解析

海洋細菌Marinomonas mediterranea由来L -リシンε-オキシダーゼの精製と性質

稲垣賢二, 赤地周作, 田村隆, 松井大亮, 岡崎誠司, 浅野泰久

2014年度ビタミンB研究委員会報告書 88 ( 9 ) 1 - 1 2015年

天野万里, 橋本義輝, 小林達彦, 近藤裕輝, 今田勝巳, 日下部均, 田村隆, 稲垣賢二

2014年度ビタミンB研究委員会報告書 89 ( 3 ) 19 - 19 2015年

3P-026 共役アッセイ法を用いたヒト肺由来セレノリン酸合成酵素の機能解析(酵素学,酵素工学,一般講演)

浅野香織, 戸部隆太, 三原久明, 根本理子, 稲垣賢二, 田村隆

日本生物工学会大会講演要旨集 67 277 - 277 2015年

2P-184 ICP-MS を用いた硫酸還元菌由来[NiFeSe]型ヒドロゲナーゼの同定(バイオマス,資源,エネルギー工学,一般講演)

三島朱加, 井本知志, 森川大地, 根本理子, 稲垣賢二, 田村隆

日本生物工学会大会講演要旨集 67 220 - 220 2015年

2-I-27 セレノリン酸合成酵素のPEPジキナーゼ共役法の確立(一般演題要旨,第66回大会講演要旨)

田村隆, 鎌田早帆, 稲垣賢二

ビタミン 88 ( 4 ) 240 - 240 2014年4月

2-IV-21 L-メチオニンγ-リアーゼ基質複合体のX線結晶構造解析と基質認識に関わる残基の機能解析(一般演題要旨,第66回大会講演要旨)

安田江里, 古谷和大, 佐藤暖, 福本充樹, 工藤大蔵, 志波智生, 原田繁春, 田村隆, 稲垣賢二

ビタミン 88 ( 4 ) 261 - 261 2014年4月

海洋細菌Marinomonas mediterranea由来L-リシンε-オキシダーゼの精製と性質検討、X線結晶構造解析

赤地周作, 岡崎誠司, 松井大亮, 田村隆, 稲垣賢二, 浅野泰久

ビタミン 88 ( 4 ) 240 - 240 2014年4月

高基質特異性L-グルタミン酸オキシダーゼより作成した基質特異性改変酵素L-アルギニンオキシダーゼの性質検討

中井隆一郎, 藤野志保子, 内海友宏, 田村隆, 日下部均, 稲垣賢二

岡山大学農学部学術報告 103 5 - 9 2014年2月

記述言語：日本語出版者・発行元：岡山大学農学部

L‒Glutamate oxidase (LGOX) from Streptomyces sp. X‒119‒6 has strict substrate specificity towardL‒glutamate. Recently, we solved the X‒ray crystal structure of LGOX and this revealed that Arg305 inthe active site is the key residue involved in substrate recognition. Therefore, we created 19 mutantenzymes of R305X‒LGOX by saturation mutagenesis. One of them R305D‒LGOX, Arg305 substitutedwith Asp exhibited oxidase activity for L‒Arg. Optimum pH of R305D‒LGOX mutant enzyme was pH8.5. Interestingly, the activity of R305D‒LGOX toward L‒Arg was inhibited by phosphate. And furthermore,the substrate specificity of R305D‒LGOX was affected by using buffer. The results of inhibitionanalysis suggest, that phosphate is a competitive inhibitor of R305D‒LGOX when L‒Arg is used assubstrate. Kinetic analysis of R305D‒LGOX showed that Km value and kcat value of R305D‒LGOX towardl-Arg were 0.68 mM and 6.7 s-1 respectively. In this study, we showed that R305D‒LGOX mutantenzyme is a novel l-arginine oxidase and useful for l-arginine biosensor.

1P-053 L-グルタミン酸オキシダーゼから作成した新規L-チロシンオキシダーゼの精製と性質(酵素学,酵素工学,一般講演)

藤野志保子, 田村隆, 日下部均, 稲垣賢二

日本生物工学会大会講演要旨集 66 30 - 30 2014年

1P-052 糸状菌由来L-リシンα-オキシダーゼの放線菌を用いた異種発現系構築(酵素学,酵素工学,一般講演)

天野万里, 水口春香, 齋藤結希, 橋本義輝, 小林達彦, 日下部均, 田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 66 30 - 30 2014年

海洋細菌Marinomonas mediterranea由来L‐リシンε‐オキシダーゼの性質検討

赤地周作, 田村隆, 松井大亮, 岡崎誠司, 浅野泰久, 稲垣賢二

生化学 85 ( 7 ) 603 2013年7月

2-II-11 共役法によるセレノリン酸合成酵素の機能解析(一般演題要旨,第65回大会講演要旨)

鎌田早帆, 田村隆, 奥河内貴大, 稲垣賢二

ビタミン 87 ( 4 ) 245 - 245 2013年4月

1-III-4 Trichoderma viride由来抗腫瘍性酵素L-Lysine α-oxidaseのX線結晶構造(一般演題要旨,第65回大会講演要旨)

天野万里, 新屋敷健悟, 佐野宰久, 川口辰也, 日下部均, 今田勝巳, 田村隆, 稲垣賢二

ビタミン 87 ( 4 ) 207 - 207 2013年4月

海洋細菌Marinomonas mediterranea由来L‐リシンε‐オキシダーゼの精製,性質検討及び結晶化

赤地周作, 松井大亮, 岡崎誠司, 浅野泰久, 田村隆, 稲垣賢二

日本農芸化学会中四国支部講演会講演要旨集 35th 27 2013年1月

1P-073 高基質特異性L-グルタミン酸オキシダーゼより作成した基質特異性改変酵素(R305D & R305L)の性質(酵素学,酵素工学,一般講演)

中井隆一郎, 藤野志保子, 田村隆, 佐野宰久, 今田勝巳, 日下部均, 稲垣賢二

日本生物工学会大会講演要旨集 65 36 - 36 2013年

2P-062 ジスルフィドイソメラーゼ活性を示すDsbA[CXXC]の機能解析(酵素学,酵素工学,一般講演)

周藤慎也, 清遠亜沙子, 稲垣賢二, 田村隆

日本生物工学会大会講演要旨集 65 119 - 119 2013年

マンガン酸化系を構成する2種の細菌の相互作用解析

田村隆, 周藤慎也, 稲垣賢二

特別経費(プロジェクト分)－大学の特性を生かした多様な学術研究機能の充実－成果報告書 89 2013年

抗腫瘍性酵素 L-リシンα-オキシダーゼのX線結晶構造解析

稲垣賢二, 天野万里, 田村隆, 佐野宰久, 今田勝己, 日下部均

ビタミン 87 ( 8 ) 463 - 464 2013年

L-グルタミン酸オキシダーゼより作成したL-ヒスチジンオキシダーゼの構造解析とL-アルギニンオキシダーゼの特性解析

稲垣賢二, 中井隆一郎, 田村隆, 今田勝巳, 日下部均

ビタミンB研究委員会報告書 87 ( 1 ) 51 - 52 2013年

2-V-16 セレノリン酸合成酵素SPS1/SPS2の相互作用(一般演題要旨,日本ビタミン学会第64回大会講演要旨)

田村隆, 奥河内貴大, 鎌田早帆, 高畑宗明, 稲垣賢二

ビタミン 86 ( 4 ) 289 - 289 2012年4月

好熱性細菌Thermus sp. O-3-1由来耐熱性アミダーゼの精製及び性質検討

小林史明, 青峰弘起, 水無渉, 湯不二夫, 田村隆, 稲垣賢二

岡山大学農学部学術報告 101 1 - 6 2012年2月

記述言語：日本語出版者・発行元：岡山大学農学部

The gene encoding a thermostable amidase (EC 3.5.1.4) from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally,as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA.好熱性細菌Thermus sp.O-3-1 由来の耐熱性アミダーゼ遺伝子を大腸菌中にクローニングし，その塩基配列を決定した．ami 遺伝子は930 bp からなり，310アミノ酸をコードしていた．本酵素の分子量は33，089 Daであると予想された．Thermus sp.O-3-1 由来アミダーゼを大腸菌で生産させ，熱処理とDEAE-トヨパール650M陰イオン交換カラム等により精製した．ゲル濾過クロマトグラフィーとSDS-PAGE の結果から本酵素は分子質量33 kDa のサブユニット2分子からなるダイマー構造を有していることが明らかとなった．精製酵素の熱安定性は80℃まで，pH 安定性は7.0～10.0であり，安定性の高い酵素であった．最適温度は90℃，最適 pH は9.0であった．EDTA により活性が著しく阻害され，Co(2+)やNi(2+)，Mn(2+)によって活性の回復，向上が見られたため，本酵素は金属酵素であることが示唆された．基質特異性の検討の結果，L-Leu-pNA よりもD-Leu-pNA に対して高い活性を示したため，本酵素がＤ-アミノ酸基質に特異性を持つアミダーゼであることが判明した．本酵素は耐熱性を有するユニークなＤ-アミノ酸アミダーゼであり，今後産業利用が期待される．

2Dp10 系統樹解析と分子モデリングに基づく[NiFeSe]型ヒドロゲナーゼの分子進化解析(分類,系統,遺伝学/遺伝子工学/植物細胞工学,組織培養,育種工学,一般講演)

田村隆, 恒川直樹, 平野敏行, 佐藤文俊, 稲垣賢二

日本生物工学会大会講演要旨集 64 52 - 52 2012年

3Cp04 高基質特異性L-グルタミン酸オキシダーゼの基質認識機構の解析及び基質特異性改変酵素の性質検討(酵素学,酵素工学,一般講演)

中井隆一郎, 田村隆, 日下部均, 今田勝巳, 稲垣賢二

日本生物工学会大会講演要旨集 64 123 - 123 2012年

L-メチオニンγ-リアーゼC116H変異酵素の構造と機能解析

福本充樹, 工藤大蔵, 原田繁春, 田村隆, 稲垣賢二

ビタミンB研究委員会報告書 85 ( 8 ) 446 - 447 2012年

2-I-28 ヒト由来セレノシステインβリアーゼ遺伝子の発現系構築と機能解析(一般演題要旨,日本ビタミン学会第63回大会講演要旨)

田村隆, 佐藤久美, 稲垣賢二

ビタミン 85 ( 4 ) 249 - 249 2011年4月

2-IV-9 好熱好酸性アーキアSulfolobus tokodaii由来シスタチオニンγ-シンターゼのPhe97残基の機能解析(一般演題要旨,日本ビタミン学会第63回大会講演要旨)

山田哲也, 福本充樹, 柳谷昌彦, 工藤大蔵, 田村隆, 倉光成紀, 稲垣賢二

ビタミン 85 ( 4 ) 247 - 247 2011年4月

放線菌Streptomyces sp. 590由来L-メチオニン脱炭酸酵素の精製および性質検討

前村知美, 内富久美子, 日下知香, 稲垣純子, 田村隆, 左右田健次, 稲垣賢二

岡山大学農学部学術報告 100 ( 100 ) 3 - 7 2011年2月

記述言語：日本語出版者・発行元：岡山大学農学部

L-Methionine decarboxylase [EC 4.1.1.57] catalyzes the decarboxylation of L-methionine and is a pyridoxal 5'-phosohate(PLP)-dependent enzyme. L-Methionine decarboxylase has been purified 630-fold by DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M and Sephacryl S-300 column chromatographies from Streptomyces sp.590. The enzyme has a dimeric structure with identical subunits of Mr 60,000. This enzyme shows optimum activity at pH7.0 and 45°C, and is stable between pH5.7 and pH9.0. L-Methionine decarboxylase has antitumor activity against RERF-LC-AI and HeLa cells. Ten N-terminal amino acid sequence of L-methionine decarboxylase was determined, and the sequence showed no homology with other reported proteins.

2Dp21 Thermus sp. O-3-1由来耐熱性アミダーゼの構造解析と含有金属分析(酵素学・酵素工学,一般講演)

放線菌由来フラビン酵素L-グルタミン酸酸化酵素のユニークな構造(研究論文紹介)

有馬二朗, Sasaki Chiduko, Sakaguchi Chika, Mizuno Hiroshi, Tamura Takashi, Kashima Akiko, Kusakabe Hitoshi, Sugio Shigetoshi, 稲垣賢二

ビタミン 85 ( 1 ) 23 - 24 2011年1月

ヒト由来セレノシステインβ-リアーゼ様タンパク質の発現, 活性確認と機能解析

田村隆, 佐藤久美, 稲垣賢二

ビタミン 85 ( 3 ) 155 - 155 2011年

食品分析用小型うま味センサーの開発

稲垣賢二, 田村隆, 中井隆一郎

うま味研究会助成金による成果報告書 2011年

小林史明, 青峰弘起, 大滝証, 尾高雅文, 養王田正文, 水無渉, 湯不二夫, 田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 63 140 - 140 2011年

3Ea13 ジスルフィドイソメラーゼの酸化還元電位を持つDsbAの活性測定(タンパク質工学/脂質工学,一般講演)

清遠亜沙子, 田村隆, 吉田隆真, 稲垣賢二

日本生物工学会大会講演要旨集 63 229 - 229 2011年

計算科学を取り入れた生体高分子の新機能解析法

田村隆

生化學 82 ( 9 ) 863 - 867 2010年9月

Production improvement of antifungal, antitrypanosomal nucleoside sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089.

Koji Fukuda, Takashi Tamura, Hideyuki Ito, Sayaka Yamamoto, Kozo Ochi, Kenji Inagaki

Journal of bioscience and bioengineering 109 ( 5 ) 459 - 65 2010年5月

記述言語：英語

Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.

DOI： 10.1016/j.jbiosc.2009.10.017

2-IV-30 キイロショウジョウバエ由来チオレドキシン還元酵素のレドックス機能解析(一般演題,日本ビタミン学会第62回大会発表要旨)

1-III-6 Pseudomonas putida由来L-メチオニン γ-リアーゼの活性中心残基への変異導入による基質特異性の改変(一般演題,日本ビタミン学会第62回大会発表要旨)

福本充樹, 工藤大蔵, 田村隆, 稲垣賢二

ビタミン 84 ( 4 ) 165 - 165 2010年4月

桑原光彦, 田村隆, 稲垣賢二

ビタミン 84 ( 4 ) 214 - 214 2010年4月

2-IV-28 放射性同位元素^<64>Cuを用いたBacillus sbutilis 168の銅代謝解析(一般演題,日本ビタミン学会第62回大会発表要旨)

田村隆, 金山洋介, 榎本秀一, 小林和夫, 小笠原直毅, 稲垣賢二

ビタミン 84 ( 4 ) 213 - 213 2010年4月

2-IV-29 セレノリン酸合成酵素SPS1はSPS2を抑制する(一般演題,日本ビタミン学会第62回大会発表要旨)

奥河内貴大, 田村隆, 高畑宗明, 三原久明, 江崎信芳, 稲垣賢二

ビタミン 84 ( 4 ) 214 - 214 2010年4月

シネフンギン生産菌Streptomyces incarnatus NRRL 8089由来アルギニン変換化合物「オルニチンラクタム」の同定

福田康二, 田村隆, 稲垣賢二

岡山大学農学部学術報告 99 ( 1 ) 1 - 5 2010年2月

記述言語：日本語出版者・発行元：岡山大学農学部

Sinefungin is a nucleoside antibiotic, in which a molecule of L-ornithine is linked to the 5' end of adenosine through a C-C bond. The antibiotic was isolated from the culture broth of Streptomyces incarnatus. For the purpose of detecting intermediate of sinefungin biosynthesis, resting cell suspensions were incubated with supplemental L-arginine, and L-ornithine. 50mM Arginine was converted to a compound X that has low polarity. 50mM ornithine was not converted and remained in reaction solution. Compound X was purified using HPLC, and analyzed using (1)H-NMR and FAB-MS. These analyses showed that a compound X is "ornithine-lactam" (Mw＝114), which has a structure of circularized ornithine. These results indicated that S. incarnatus has an enzyme that converts arginine to ornithine-lactam. Such an enzyme has never been reported, and suggested that it may be relevant to sinefungin biosynthesis.シネフンギンは抗真菌，抗マラリア活性を有する核酸系抗生物質であり，放線菌 S. incarnatus により生合成される．シネフンギンはアデノシンとオルニチンがＣﾝＣ結合した構造であり，無細胞抽出液での取り込み実験からＬﾝアルギニンと ATP から生合成されると推測される．Ｌﾝアルギニン，Ｌﾝオルニチンを S. incarnatus の休止菌体反応系への投与を行いシネフンギン中間体の探索を行った．その結果50ﾋアルギニンは24時間以内に低極性化合物へと変換された．一方50ﾋオルニチンは変換されず反応液中に残存した．HPLC で化合物を精製し，1HﾝNMR，FABﾝMS での分析の結果オルニチン環状モノペプチド，「オルニチンラクタム」（分子量114）であることを明らかにした．この結果は S. incarnatus がアルギニンからオルニチンラクタムへの変換酵素を有する事を示唆する．このような酵素の報告例はこれまでになく，ニ次代謝酵素であることが示唆され，シネフンギン生合成との関連性に興味が持たれる．

好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニン γ-シンターゼの精製及び性質検討

篠崎舞, 柳谷昌彦, 兼田翔一郎, 工藤大蔵, 遠藤祐一, 田村隆, 倉光成紀, 稲垣賢二

岡山大学農学部学術報告 99 ( 1 ) 7 - 12 2010年2月

記述言語：日本語出版者・発行元：岡山大学農学部

The gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase ［EC 2. 5. 1. 48］ from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine.好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニンγﾝシンターゼ（stCGS）遺伝子を pET-11a に組み込み pET-stCGS を構築した．このベクターでE. coli Rosettaﾝgami（DE3）を形質転換し，本遺伝子を発現させ，精製及び性質検討を行った．大腸菌で発現したシスタチオニンγﾝシンターゼの活性が無細胞抽出液で確認できた．S. tokodaii シスタチオニンγﾝシンターゼを70℃熱処理 DEAEﾝトヨパールイオン交換カラム等により単一精製した．精製酵素の最適温度は100℃以上であり，熱安定性は60分間処理で70℃までほぼ100ｵの残存活性を示した．また，最適pHについてはリン酸緩衝液やブリトンﾝロビンソン広域緩衝液の場合はpH7.0の時が最も活性が高く，トリス塩酸緩衝液の場合はpH9.0が最適であった．pH安定性についてはpH5.0～9.0において安定であった．O-ホスホ-l-ホモセリンに対するKm，Vmaxは，それぞれ0.82mM，2.42U/㎎であった．アポ酵素のホロ化実験により，本酵素活性がPLP に依存していることが明らかとなった．更に本酵素の脱離反応での基質特異性の検討を行った．変異酵素を用いた実験により，stCGSの基質特異性には，活性中心に存在するPhe97を含む領域が深く関わっていることが示唆された．

3P-1075 Trichoderma viride由来抗腫瘍性酵素L-リシンα-オキシダーゼの精製と結晶化(2a酵素学,酵素工学,一般講演,酵素学,タンパク質工学および酵素工学,伝統の技と先端科学技術の融合)

中性脂肪測定用酵素グリセロールキナーゼの構造特性解析と耐熱化

稲垣賢二, 福田靖久, 田村隆, 岸本高英, 曽我部敦

2009年度ビタミンB研究委員会報告書 83 ( 11 ) 632 - 633 2010年

新屋敷健悟, 中田春香, 今田勝巳, 田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 22 76 - 76 2010年

2P-1053 ジスルフィドイソメラーゼの酸化還元電位を持つDsbA[CRIC]の発現系検討(2a酸素学,酵素工学,一般講演,酵素学,タンパク質工学および酵素工学,伝統の技と先端科学技術の融合)

清遠亜沙子, 田村隆, 吉田隆真, 稲垣賢二

日本生物工学会大会講演要旨集 22 22 - 22 2010年

Structural characterization of l-glutamate oxidase from Streptomyces sp X-119-6

Jiro Arima, Chiduko Sasaki, Chika Sakaguchi, Hiroshi Mizuno, Takashi Tamura, Akiko Kashima, Hitoshi Kusakabe, Shigetoshi Sugio, Kenji Inagaki

FEBS JOURNAL 276 ( 14 ) 4318 - 4327 2009年7月

記述言語：英語出版者・発行元：WILEY-BLACKWELL

L-Glutamate oxidase (LGOX) from Streptomyces sp. X- 119- 6, which catalyzes the oxidative deamination of L-glutamate, has attracted increasing attention as a component of amperometric L-glutamate sensors used in the food industry and clinical biochemistry. The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an alpha(2)beta(2)gamma(2) structure; enzymatic proteolysis of the precursor forms the stable mature enzyme. We solved the crystal structure of mature LGOX using molecular replacement with a structurally homologous model of l- amino acid oxidase ( LAAO) from snake venom: LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of LAAO revealed that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. Furthermore, the arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO. Results of the comparative analyses described herein raise the possibility that such a unique structure of LGOX is associated with its substrate specificity.

DOI： 10.1111/j.1742-4658.2009.07103.x

2-I-16 亜セレン酸の還元代謝に関わるセレン-Thioredoxin付加体の同定(一般研究発表,日本ビタミン学会第61回大会研究発表要旨)

佐藤久美, 田村隆, 古森健太郎, 戸部隆太, 三原久明, 江崎信芳, 稲垣賢二

ビタミン 83 ( 4 ) 191 - 191 2009年4月

2-II-24 放線菌由来L-グルタミン酸オキシダーゼの構造特性と基質認識機構の解析(一般研究発表,日本ビタミン学会第61回大会研究発表要旨)

内海友宏, 田村隆, 坂口智香, 有馬二郎, 日下部均, 稲垣賢二

ビタミン 83 ( 4 ) 208 - 208 2009年4月

高度好熱性細菌Thermus thermophilus HB8由来アラニンラセマーゼの大腸菌での発現, 精製及び諸性質の検討

柳谷昌彦, 上前智, 白神智行, 田村隆, 稲垣賢二

岡山大学農学部学術報告 98 ( 1 ) 1 - 7 2009年2月

記述言語：日本語出版者・発行元：岡山大学農学部

An alanine racemase (EC 5.1.1.1) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 aminoacid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0∼10.0 and an optimum temperature of 55°C∼60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa.高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼ遺伝子を大腸菌中にクローニングし、発現させた後に、精製及び性質検討を行った。alr遺伝子は1080bpからなり360アミノ酸残基 HB8をコードしていたので、本酵素は38,596Daの分子量であると予想された。alr遺伝子の [ G + C ] 含量は、72%であり、Tm値は98.8℃であった。T.thermophilus HB8由来アラニンセマーゼを中等度好熱性細菌 Geobacillus stearothermophilus 及び赤痢菌 Shigella sonnei 由来アラニンラセマーゼと一次配列の比較をしたところ、G.stearothermophilus 由来のものと33%、赤痢菌由来の酵素を28%の相同性を示した。T.thermophilus HB8 由来アラニンラセマーゼを、70℃で10分間の熱処理後、DEAE-トーヨーパール陰イオン交換カラム等により精製した。精製酵素の最適温度は、d-アラニンからl-アラニンへの反応で55℃、l-アラニンからd-アラニンへの反応では60℃であり、最適pHは、9.0〜10.0であった。また、70℃で30分インキュベーションを行った後にも、活性の低下は見受けられず耐熱性を示した。更に、本酵素は分子量38,000モノマー酵素であると推定され、その反応機構に興味が持たれる。

Cuckoo Fungus Mimics Termite Eggs by Producing the Cellulose-Digesting Enzyme beta-Glucosidase

Kenji Matsuura, Toshihisa Yashiro, Ken Shimizu, Shingo Tatsumi, Takashi Tamura

CURRENT BIOLOGY 19 ( 1 ) 30 - 36 2009年1月

記述言語：英語出版者・発行元：CELL PRESS

Insects and fungi share a long history of association in various habitats, including the wood-decomposition niche [7]. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect-fungus association [2, 3]. Termites of the genus Reticulitermes often harbor fungal sclerotia, called "termite balls," along with eggs in nursery chambers, whereby the fungus gains a competitor-free habitat in termite nests. Sophisticated morphological and chemical camouflage are needed for the fungus to mimic termite eggs [3]. However, the mechanism of chemical egg mimicry by the fungus is unknown. Here, we show that the fungus mimics termite eggs chemically by producing the cellulose-digesting enzyme beta-glucosidase. We found that the termite egg-recognition pheromone consists of beta-glucosidase and lysozyme. Both enzymes are major salivary compounds in termites and are also produced in termite eggs. Termite balls were tended by termites only when the fungus produced beta-glucosidase. Our results demonstrated that the overlap of the cellulose digestion niche between termites and the fungus sharing the same chemicals provided the opportunity for the origin of termite egg mimicry by the fungus. This suggests that pheromone compounds might have originally evolved within other life history contexts, only later gaining function in chemical communication.

DOI： 10.1016/j.cub.2008.11.030

2Dp12 新規耐熱性アミダーゼの遺伝子クローニングと酵素精製(酵素学・酵素工学,一般講演)

Enhanced Production of the Fluorinated Nucleoside Antibiotic Nucleocidin by a rifR-Resistant Mutant of Streptomyces calvus IFO13200

Koji Fukuda, Takashi Tamura, Yuko Segawa, Yuta Mutaguchi, Kenji Inagaki

Actinomycetologica 23 ( 2 ) 51 - 55 2009年

微生物の水素発酵に関わるギ酸脱水素酵素の発現制御機構の解明

田村隆, 稲垣賢二

岡山大学大学院自然科学研究科プロジェクト「CO2循環・削減型社会実現に向けての融合的研究」2008年度成果報告書 13 - 16 2009年

Conflicting Two Hypotheses on Reductive Selenite Assimilation in Escharichia coli

Takashi Tamura

Biomed. Res. Trace Elements 20 ( 3 ) 226 - 231 2009年

小林史明, 青峰弘起, 水無渉, 中村哲二, 湯不二夫, 田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 21 50 - 50 2009年

2Dp06 新規な活性中心配列を持つDsbA[CDIC]のジスルフィドイソメラーゼ活性(酵素学・酵素工学,一般講演)

清遠亜沙子, 田村隆, 吉田隆真, 稲垣賢二

日本生物工学会大会講演要旨集 21 48 - 48 2009年

1Bp22 rpoB遺伝子改変によるStreptomyces incarnatusのシネフンギン高生産株(遺伝子工学,一般講演)

山城大地, 福田康二, 田村隆, 越智幸三, 稲垣賢二

日本生物工学会大会講演要旨集 21 11 - 11 2009年

糸状菌Trichoderma viride由来L-リジンα-オキシダーゼ完全長cDNAの単離と大腸菌発現系構築

中田春香, 田村隆, 稲垣純子, 日下部均, 稲垣賢二

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 81回・31回 1T8 - 3 2008年11月

The role of cysteine 116 in the active site of the antitumor enzyme L-methionine gamma-lyase from Pseudomonas putida

Daizou Kudou, Shintaro Misaki, Masao Yamashita, Takashi Tamura, Nobuyoshi Esaki, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 ( 7 ) 1722 - 1730 2008年7月

記述言語：英語出版者・発行元：TAYLOR & FRANCIS LTD

The cysteinyl residue at the active site Of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.

DOI： 10.1271/bbb.80015

2-III-26 好熱好酸性アーキアSulfolobus tokodaii strain 7由来シスタチオニンγシンターゼの機能解析(一般研究発表,新世紀ビタミン学の展望と先進的展開を目指して,日本ビタミン学会第60回大会発表要旨)

亜セレン酸還元代謝の「定説」を疑う!!

田村隆, 高畑宗明, 古森健太郎, 三原久明, 江崎信芳, 稲垣賢二

Biomedical research on trace elements 19 ( 2 ) 164 - 164 2008年6月

工藤大蔵, 柳谷昌彦, 田村隆, 倉光成紀, 稲垣賢二

ビタミン 82 ( 4 ) 291 - 291 2008年4月

2-IV-16 ヒト肺由来セレノリン酸合成酵素アイソザイムのドメイン機能解析(一般研究発表,新世紀ビタミン学の展望と先進的展開を目指して,日本ビタミン学会第60回大会発表要旨)

田村隆, 高畑宗明, 三原久明, 江崎信芳, 稲垣賢二

ビタミン 82 ( 4 ) 301 - 301 2008年4月

2-IV-14 Drosophila TrxRのC末端レドックスモチーフの同定(一般研究発表,新世紀ビタミン学の展望と先進的展開を目指して,日本ビタミン学会第60回大会発表要旨)

桑原光彦, 田村隆, 稲垣賢二

ビタミン 82 ( 4 ) 300 - 300 2008年4月

Selenite assimilation into formate dehydrogenase H depends on thioredoxin reductase in Escherichia coli

Muneaki Takahata, Takashi Tamura, Katsumasa Abe, Hisaaki Mihara, Suguru Kurokawa, Yoshihiro Yamamoto, Ryuhei Nakano, Nobuyoshi Esaki, Kenji Inagaki

JOURNAL OF BIOCHEMISTRY 143 ( 4 ) 467 - 473 2008年4月

記述言語：英語出版者・発行元：OXFORD UNIV PRESS

Escherichia coli growing under anaerobic conditions produce H(2) and CO(2) by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDH(H)), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma(54)-dependent transcriptional activator Fh1A that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDHH activity, we have identified a low-salt nutrient medium containing <0.1% NaCl enabled constitutive, high expression of FDH(H) even without formate and D-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDH(H) activity and also reductive assimilation of selenite (SeO(3)(2-)) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDH(H) production and for labelling the FDH(H) polypeptide with (75)Se-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

DOI： 10.1093/jb/mvm247

海産性好熱性細菌Rhodothermus marinus由来イソアミラーゼの精製,性質検討及びX線結晶構造解析

立花亜紀子, 田村隆, 今田勝巳, 木下実紀, 難波啓一, 堤紀子, 橋田みよ子, 坂口博脩, 稲垣賢二

岡山大学農学部学術報告 97 ( 97 ) 1 - 7 2008年2月

記述言語：日本語出版者・発行元：岡山大学農学部

The isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coliTop 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimumpH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinusisoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. Theseresults suggest that R. marinus isoamylase has high thermostability. The crystallization and crystalstructure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9Åresolution was determined in complex with the panose. R. marinus isoamylase is composed of threedomains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of theR. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-siteconsensus regions characteristic of the α-amylase family. And the essential residue of the α-amylasefamily (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loopsthan P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These resultsare thought to be factors of thermostability of R. marinus isoamylase.Rhodothermus marinus 由来イソアミラーゼ遺伝子を組み込んだプラスミド pBX２を使用し，大腸菌 Top10株を形質転換し，16時間の前培養，24時間の本培養後，菌体破砕し，得られた無細胞抽出液を熱処理（80℃，10 min），50ｵ硫安分画，陰イオン交換カラムクロマトグラフィー（DEAEﾝトヨパール），ハイドロキシアパタイトカラムクロマトグラフィーに供して本酵素の精製を行った．本精製酵素の性質検討を行った結果，本酵素の最適反応温度は70℃，pH４であり，また本酵素は60℃で１時間処理しても活性が低下することが無く，Pseudomonas amyloderamosa 由来イソアミラーゼよりも高い耐熱性を有することが判明した．本酵素の結晶化・X線結晶構造解析を行った結果，本酵素は P. amyloderamosa 由来イソアミラーゼと同様Ｎドメイン・ＡドメインＣドメインの３つのドメインから構成されており，活性残基（Ｄ359，Ｅ395，Ｄ467）など活性中心付近のアミノ酸残基も P. amyloderamosa 由来イソアミラーゼと同様，高度に保存されていた．本酵素の熱安定性が P. amyloderamosa 由来イソアミラーゼよりも高い要因として，P. amyloderamosa 由来イソアミラーゼよりもループの長さが全体的に短いことと，カルシウムイオン結合サイトの欠如が挙げられた．今後さらに構造解析を進めることにより，本酵素の熱安定性機構，反応機構など更なる知見が得られることが期待される．

Structure and quantum chemical analysis of NAD(+)-dependent isocitrate dehydrogenase: Hydride transfer and co-factor specificity

Katsumi Imada, Takashi Tamura, Ryo Takenaka, Issei Kobayashi, Keiichi Namba, Kenji Inagaki

PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 70 ( 1 ) 63 - 71 2008年1月

記述言語：英語出版者・発行元：WILEY-LISS

The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD(+) and citrate has been solved to a resolution of 1.9 angstrom. The protein fold of this NAD(+)-dependent enzyme shares a high similarity with those of NADP(+-)dependent bacterial 1CDHs. The NAD(+) and the citrate are clearly identified in the active site cleft with a well-defined electron density. Asp-357 is the direct cofactor-specificity determinant that interacts with 2'-OH and 3'-OH of the adenosine ribose. The adenosine ribose takes a C2'-endo puckering conformation as previously reported for an NAD(+-)specific isopropylmalate dehydrogenase. The nicotinamide moiety of NAD(+) has the amide NH2 group oriented in cis conformation with respect to the C4 carbon of the nicotinamide ring, slanted toward the bound citrate molecule with a dihedral angle of -21 degrees. The semi-empirical molecular orbital calculation suggests that the pro-R hydrogen atom at C4 of NADH would bear the largest negative charge when the amide NH2 group is in such conformation, suggesting that the amide group has a catalytically significant role in stabilizing the transition state as NADH is being formed during the hydride transter catalysis.

DOI： 10.1002/prot.21486

1Fp11 祖先型変異導入によるグリセロールキナーゼの耐熱化(酵素学,酵素工学,タンパク質工学,一般講演)

2Fa07 糸状菌Trichoderma viride由来の抗腫瘍性酵素L-リジンα-オキシダーゼ : 遺伝子の単離と解析(酵素学,酵素工学,タンパク質工学,一般講演)

中田春香, 田村隆, 稲垣純子, 日下部均, 稲垣賢二

日本生物工学会大会講演要旨集 20 159 - 159 2008年

臨床診断・生化学検査に貢献する酵素の構造と機能解析

田村隆, 稲垣賢二

生物工学会誌 86 ( 4 ) 177 - 179 2008年

福田靖久, 田村隆, 阿部明香, 岸本高英, 曽我部敦, 稲垣賢二

日本生物工学会大会講演要旨集 20 155 - 155 2008年

The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

Kenji Matsuura, Takashi Tamura, Norimasa Kobayashi, Toshihisa Yashiro, Shingo Tatsumi

PLOS ONE 2 ( 8 ) 813 2007年8月

記述言語：英語出版者・発行元：PUBLIC LIBRARY SCIENCE

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites.

DOI： 10.1371/journal.pone.0000813

2-II-7 Q494R型チオレドキシン還元酵素の基質特異性(ビタミン学の原点・栄養学への21世紀的回帰, 日本ビタミン学会第59回大会)

桑原光彦, 田村隆, 奥山祐子, 稲垣賢二

ビタミン 81 ( 4 ) 154 - 154 2007年4月

2-II-6 分子軌道計算に基づくTrxRの精密分子設計とアレニウスプロット解析(ビタミン学の原点・栄養学への21世紀的回帰, 日本ビタミン学会第59回大会)

田村隆, 奥山祐子, 中山恵美, 稲垣賢二

ビタミン 81 ( 4 ) 153 - 153 2007年4月

抗腫瘍性酵素L-リジンα-オキシダーゼの遺伝子クローニング

中田春香, 田村隆, 稲垣純子, 日下部均, 稲垣賢二

ビタミン 81 ( 4 ) 148 - 148 2007年4月

Structure of the antitumour enzyme L-methionine γ-lyase from Pseudomonas putida at 1.8 A Resolution

D. Kudou, S. Misaki, M. Yamashita, T. Tamura, T. Takakura, T. Yoshioka, S. Yagi, R. M. Hoffman, A. Takimoto, N. Esaki, K. Inagaki

J. Biochem 141 ( 4 ) 535 - 544 2007年4月

キイロショウジョウバエ由来のチオレドキシン還元酵素のC末端テトラペプチド配列は、ヒト肺由来のチオレドキシン還元酵素では酸化還元活性を示さない

桑原光彦, 田村隆, 河村健太郎, 稲垣賢二

岡山大學農學部學術報告 = Scientific report of the Faculty of Agriculture, Okayama University ( 96 ) 1 - 5 2007年2月

記述言語：日本語出版者・発行元：岡山大學農學部

ほ乳類チオレドキシン還元酵素はC末端配列-Gly-Cys-SeCys-Gly（end）の後ろから2番目にセレノシステイン（SeCys）残基を持つ。SeCysをシステインに変換すると酵素の活性は大きく低下するので、SeCys残基が触媒活性に必須であることが分かる。これに対してキイロショウジョウバエのチオレドキシン還元酵素（Dm-rxR）のC末端配列にはセレンが含まれず、システイン残基の対が2つのセリンに挟まれた配列-Ser-Cys-Cys-Ser（end）を持つ。それでもDm-rxRはほ乳類のセレン含有酵素と同程度の触媒能を示す。われわれはヒト肺チオレドキシン還元酵素にDm-rxRのC末端テトラペプチド配列を導入してその効果を調べた。しかし、酵素活性はまったく上昇せず、Dm-rxRのC末端のテトラペプチド配列-Ser-Cys-Cys-SerだけではCys残基のチオール基を活性化する効果はなかった。そこで、分子軌道計算MOPACを用いて酸化還元機能を担うためのC末端配列モチーフを探索した。その結果、テトラペプチドにさらに2つ先のプロリンまでを含めたPro-X-Ser-Cys-Cys-Ser（end）により初めて酸化還元モチーフとして機能する可能性が示唆された。Proを含むこの配列モチーフはミツバチや蚊などほかの昆虫のrxRでも保存されていた。

1C11-4 L-グルタミン酸オキシダーゼの立体構造と機能の相関解析(酵素学・酵素工学・タンパク質工学,一般講演)

The C-terminal Tetrapeptide Sequence of Drosophila Thioredoxin Reductase Does not Function as a Redox-active Motif in the Human Lung Counterpart

Kuwahara, M, Tamura, T, Kawamura, K, Inagaki, K

Sci. Rep. Fac. Agric. Okayama Univ. 96 1-5 1 - 5 2007年

大腸菌の亜セレン酸代謝はチオレドキシン還元酵素に依存する

高畑宗明, 田村隆, 阿部勝正, 三原久明, 江崎信芳, T.C. Stadtman, 稲垣賢二

Trace Nutrient Res., 24, 42-48 (2007) 24 42 - 48 2007年

有馬二朗, 佐々木千津子, 坂口智香, 田村隆, 日下部均, 杉尾成俊, 稲垣賢二

日本生物工学会大会講演要旨集 19 79 - 79 2007年

2S3PM5 臨床診断・生化学検査に貢献する酵素の構造と機能解析(バイオテクノロジーへの応用的戦略を踏まえたタンパク質構造解析,シンポジウム)

田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 19 34 - 34 2007年

2B09-2 超好熱好酸性古細菌Sulfolobus tokodaii strain 7由来ホモセリンキナーゼの大腸菌での発現と性質検討(酵素学・酵素工学・タンパク質工学,一般講演)

柳谷昌彦, 工藤大蔵, 倉光成紀, 田村隆, 稲垣賢二

日本生物工学会大会講演要旨集 19 61 - 61 2007年

1C11-3 抗腫瘍性酵素L-メチオニンγ-リアーゼの変異酵素C49Kの作製及び性質検討(酵素学・酵素工学・タンパク質工学,一般講演)

工藤大蔵, 田村隆, 瀧本明生, 高倉知朗, 稲垣賢二

日本生物工学会大会講演要旨集 19 79 - 79 2007年

Performance characterization of recombinant L-glutamate oxidase in a micro GOT/GPT sensing system

Sanjay Upadhyay, Naoto Ohgami, Hitoshi Kusakabe, Hiroshi Mizuno, Jiro Arima, Takashi Tamura, Kenji Inagaki, Hiroaki Suzuki

SENSORS AND ACTUATORS B-CHEMICAL 119 ( 2 ) 570 - 576 2006年12月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE SA

Large-scale production Of L-glutamate oxidase (L-GlOx) from Streptomyces sp. X-119-6 was conducted with the use of an Escherichia coli expression system. The active alpha(2)beta(2)gamma(2)-subunit structure of the enzyme was obtained by proteolysis of a precursor form using metalloendopeptidase. The performance of this recombinant enzyme was tested in an amperometric sensing system to determine the L-glutamate concentration. To this end, a thin-film, three-electrode system was formed. Hydrogen peroxide produced by an enzymatic reaction was detected by using a platinum working electrode. The sensitivity of the L-glutamate sensor was 220 nA/mM, and the lower detection limit was 3 mu M (S/N = 3). Up to 800 mu M, a linear relationship was observed between the output current and the L-glutamate concentration. Furthermore, we used a microanalysis system to determine the activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) and, ultimately, to test whether the recombinant enzyme can additionally be used to analyze enzyme activities. A polydimethylsiloxane (PDMS) flow channel with a mixing compartment was also used. The substrate solution and the sample solution for either GOT or GPT were mixed as they were being removed from the flow channel. As time elapsed, the L-glutamate concentration in the mixed solution increased because of the enzymatic reaction of GOT or GPT; consequently, a constant increase in current was observed. The relation between the slope of the response curve and the enzyme activity was linear up to 127 U/l for GOT and to 88 U/l for GPT. The results of this series of experiments demonstrate that recombinant L-GlOx can be used to construct micro sensors and microanalysis systems. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2006.01.008

2-III-25 放線菌由来L-グルタミン酸オキシダーゼの大腸菌での大量精製系の確立(一般研究発表,日本ビタミン学会第58回大会研究発表要旨)

The crystal structure of hypothetical methyltransferase from Thermus thermophilus HB8

C Sasaki, Sugiura, I, A Ebihara, T Tamura, S Sugio, K Nagaki

PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 64 ( 2 ) 552 - 557 2006年8月

坂口智香, 水野寛士, 田村隆, 日下部均, 稲垣賢二

ビタミン 80 ( 4 ) 253 - 253 2006年4月

1-II-6 抗腫瘍性酵素L-methionine γ-lyaseの活性中心におけるAsp241の機能解析(一般研究発表,日本ビタミン学会第58回大会研究発表要旨)

工藤大蔵, 岬真太郎, 瀧本明生, 田村隆, 稲垣賢二

ビタミン 80 ( 4 ) 208 - 208 2006年4月

2-III-14 大腸菌の亜セレン酸同化はチオレドキシン還元酵素に依存する(一般研究発表,日本ビタミン学会第58回大会研究発表要旨)

田村隆, 高畑宗明, 阿部勝正, 三原久明, 江崎信芳, 稲垣賢二

ビタミン 80 ( 4 ) 247 - 247 2006年4月

Characterization of monofunctional catalase KatA from radioresistant bacterium Deinococcus radiodurans

Kobayashi, I, T Tamura, H Sghaier, Narumi, I, S Yamaguchi, K Umeda, K Inagaki

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 101 ( 4 ) 315 - 321 2006年4月

記述言語：英語出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we report on the cloning, purification and characterization of a catalase (KatA, DR1998) from the extremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa, suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide, sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret band at 408 nm. The position of the spectral peak remained unchanged following reduction of KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance property may play a role in the survival strategy of D. radiodurans against oxidative damage.

DOI： 10.1263/jbb.101.315

染料脱色微生物による染料脱色機構の解析

馬場直子, 坂口博脩, 田口隆章, 早瀬伸樹, 田村隆, 稲垣賢二

岡山大学農学部学術報告 95 ( 95 ) 1 - 5 2006年2月

記述言語：日本語出版者・発行元：岡山大学農学部

The mehcanism of azo dyes decolorization by Candida sp. MK-1, Aeromonas sp. B-5 and Actinobacillus sp. B-11 were analyzed. The maximal decolorization activity was observed at pH 7.5 and 30℃ on Candida sp. MK-1, at alkaline and at 35℃ on Aeromonas sp. B-5 and Actinobacillus sp. B-11. The HPLC analysis of the supernatant of the Acid Red 27 detected in the blank. The retention time of this peak matched that of a reference standard compound of 4-amino-1-naphthalenesulfonate, produced by reductive cleavage of Acid Red 27. The decolorization of azo dyes with cell free extract of Candida sp. MK-a was promoted by the addition of several coenzymes or lawsone. The remarkable promotion of decolorization was observed by the addition of glutamate dehydrogenase with α-ketoglutarate and NH4+. Therefore, it was suggested that Candida sp. MK-1 azoreductase catalyzed decolorization of azo dye by NADPH dependent reductive cleavage.

1F10-2 放線菌Streptomyces avermitilis由来L-メチオニンγリアーゼの精製と性質検討(酵素学,酵素工学,タンパク質工学,一般講演)

稲垣賢二, 工藤大蔵, 平井祥之, 田村隆

日本生物工学会大会講演要旨集 18 92 - 92 2006年

2-B-13 NAD依存型イソクエン酸脱水素酵素の量子酵素化学(第57回大会一般研究発表)(第57回大会研究発表要旨)

田村隆, 今田勝巳, 竹中涼, 小林一聖, 稲垣賢二

ビタミン 79 ( 4 ) 251 - 251 2005年4月

化学合成独立栄養細菌Acidithiobacillus thiooxidans 由来アコニターゼの遺伝子解析と大腸菌での発現

金原陽平, 田村隆, 徳田千束, 中村淳雄, 松川寛和, 稲垣賢二

岡山大学農学部学術報告 94 1 - 7 2005年

記述言語：日本語出版者・発行元：岡山大學農學部

Acidihiobacillus hiooxidans由来アコニターゼ遺伝子を大腸菌中にクローニングし、発現させ、精製及び性質検討を行った。A. hiooxidansアコニターゼはEscherichia coli JM109株で発現させ、活性が無細胞抽出液で確認された。646アミノ酸から成っており、イソクエン酸脱水素酵素遺伝子（icd）の上流に位置していた。またブタ心筋由来アコニターゼと大腸菌アコニターゼA型と高いアミノ酸配列相同性があり、ブタ心筋アコニターゼで触媒反応、基質の結合に関与するとされている27アミノ酸のうち25アミノ酸の保存が確認された。A. hiooxidansアコニターゼはDEAE-oyopearl 650M等により精製された。精製酵素は至適pH7.5、至適温度60℃であった。また40℃で1時間インキュベートを行った後にも、活性の低下は見受けられなかった。pH6.0-8.0で1時間インキュベートした後も、100％の活性を維持していた。更に本酵素は、分子量66000のモノマー酵素であることが明らかになった。なお、本論文中に記載したA. hiooxidansのアコニターゼ遺伝子を含む塩基配列はDDBJデータベースに登録されており、アクセション番号AB196983で検策できる。

ヒト肺がん細胞のセレノリン酸合成酵素Sps1,Sps2の機能解析

その他リンク： http://agriknowledge.affrc.go.jp/RN/2010710742

田村隆, 山本真平, 高畑宗明, 田中英彦, 稲垣賢二

微量栄養素研究 22 147 - 152 2005年

バイオファクター・セレンを含む新規酵素の発見と肺がん細胞のセレン代謝特性の解明

田村隆

ビタミン 79 ( 3 ) 143 - 153 2005年

記述言語：日本語出版者・発行元：公益社団法人日本ビタミン学会

Selenium is an essential trace element for mammals, birds, and some bacteria. Its remarkable biological effects in eukaryotes may be related to unique functions of various selenoproteins. A^<75>Se-labeled protein was purified from a human lung adenocarcinoma cell line, NCI-H441 . A homodimer of 57-kDa subunits contained selenium in the form of selenocysteine. The selenoprotein contained FAD as a prosthetic group and catalyzes NADPH-dependent reduction of insulin in the presence of thioredoxin. The subunit composition and catalytic properties were identical to those of mammalian thioredoxin reductase (TrxR). This was the first report on the biochemical relevance between selenium and Trx-mediated metabolic regulation. Interestingly, human lung adenocarcinoma cells produced the selenoenzyme TrxR over hundred times that of normal mammalian cells. A Iabile selenium donor compound monoselenophosphate, required for selenoprotein biosynthesis, is synthesized from selenide and ATP by selenophosphate synthetase (SPS).

DOI： 10.20632/vso.79.3_143

2C13-5 放線菌由来のバイオセンサー用酵素L-グルタミン酸オキシダーゼの大腸菌での精製系確立とX線結晶構造解析(酵素学・酵素工学・タンパク質工学,一般講演)

その他リンク： http://search.jamas.or.jp/link/ui/2005136810

稲垣賢二, 坂口智香, 田村隆, 日下部均, 鹿島亜希子, 佐々木千津子, 杉尾成俊

日本生物工学会大会講演要旨集 17 109 - 109 2005年

Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation

T Tamura, S Yamamoto, M Takahata, H Sakaguchi, H Tanaka, TC Stadtman, K Inagaki

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 ( 46 ) 16162 - 16167 2004年11月

記述言語：英語出版者・発行元：NATL ACAD SCIENCES

A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCI-H441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Spsi or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added L-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles L-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.

DOI： 10.1073/pnas.0406313101

Saccharomyces cerevisiae 由来D-アミノ酸アセチルトランスフェラーゼの精製と性質.

2-II-26 超好熱好酸性古細菌Sulfolobus tokodaii由来シスタチオニンγ-シンターゼの精製および性質検討(第56回大会一般研究発表)

遠藤祐一, 田村隆, 倉光成紀, 田中英彦, 稲垣賢二

ビタミン 78 ( 4 ) 246 - 246 2004年4月

田代(山田, 百合子, 田村隆, 田中英彦, 稲垣賢二

岡山大学農学部学術報告 93 ( 1 ) 13 - 18 2004年

記述言語：日本語出版者・発行元：岡山大学農学部

Some properties of D-amino scid acetyltransferase purified from Saccharomyces cerevisiae were investigated. The enzyme was purified to homogeneity by ammonium sulfate fractionation, column chromatographies on DEAE-Toyopearl 650M, Sephacryl S-200, QAE-Toyopearl 550C and affinity chromatography with D-glutamate as a ligand. The molecular weight was estimated to be about 53,000 by gel filtration. Relative molecular mass studies indicated that the enzyme was a monomer structure. The purified enzyme had an optimum pH of 8.4 and an optimum temperature of 40C. The Km values of the purified enzyme determined with tryptophan and acetyl-CoA were 4.5 * 10 -3M, respectively. The 20 residues of N-terminal amino acid sequence were analyzed.

2A11-4 DsbAコンビナトリアル変異ライブラリーの酸化還元電位評価(酵素学・酵素工学・タンパク質工学,一般講演)

田村隆, 植村祐子, 永安牧子, 稲垣賢二

日本生物工学会大会講演要旨集 16 106 - 106 2004年

Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp X-119-6

J Arima, T Tamura, H Kusakabe, M Ashiuchi, T Yagi, H Tanaka, K Inagaki

JOURNAL OF BIOCHEMISTRY 134 ( 6 ) 805 - 812 2003年12月

記述言語：英語出版者・発行元：JAPANESE BIOCHEMICAL SOC

L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha(2)beta(2)gamma(2). The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha(2)beta(2)gamma(2) and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.

DOI： 10.1093/jb/mvg206

Cosynthesis of monofluoroacetate and 4-fluorothreonine by resting cells of blocked mutants of Streptomyces cattleya NRRL8057

T Tamura, Y Sawamoto, T Kuriyama, K Oba, H Tanaka, K Inagaki

JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 23 ( 2-6 ) 257 - 263 2003年9月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE BV

Streptomyces cattleya NRRL 8057 produces monofluoroacetate and 4-fluorothreonine from inorganic fluoride. Mutants blocked in fluorometabolite production were prepared by chemical mutagenesis, and cosynthesis experiments with these blocked mutants were carried out by suspending cells of one blocked mutant in the supernatant broth of another blocked mutant. The harvest age of the cells, pH of the buffer, potassium fluoride concentration and glycerol supplementation were optimized for the monofluoroacetate production by a resting-cell suspension of S. cattleya. Successful cosynthesis with pairs of the mutants characterized four distinctive blocked sites in the order N-82, N-44, N-43 and N-47. Additional preparation of blocked mutants by UV irradiation and their cosynthesis assay confirmed that U-303, U-304, U-400 and U-500 were blocked in later steps than N-47. O'Hagan et al. recently proposed that fluoroacetaldehyde, the hypothetical precursor of monofluoroacetate and 4-fluorothreonine, derives from 5'-fluoro-5-deoxyadenosine, the first fluorinated metabolite synthesized from S-adenoSyl-L-methionine and inorganic fluoride by the novel enzyme 'fluorinase'. We were able to detect fluorinase activity in crude extracts of wild type and N-47 mutant strains, but not in the other mutant strains whose blocked steps flanked that of N-47. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00088-2

ヒト由来チオレドキシン還元酵素のカルボキシル末端配列の酸化還元状態の半経験的分子軌道計算

Purification and substrate characterization of α-ketobutyrate decarboxylase from Pseudomonas putida

Hiroyuki Inoue, Akira Nishito, Shin-ich Eriguchi, Takashi Tamura, Kenji Inagaki, Hidehiko Tanaka

J. Mol. Catal. B: Enzym. 23 ( 2-6 ) 265 - 271 2003年9月

田村隆, 田中英彦, 稲垣賢二

岡山大學農學部學術報告 = Scientific report of the Faculty of Agriculture, Okayama University ( 92 ) 17 - 20 2003年2月

記述言語：日本語出版者・発行元：岡山大學農學部

ヒトのチオレドキシン還元酵素（TrxR）のカルボキシル末端配列の酸化還元に伴うエンタルピー変化を計算した。半経験的分子軌道計算WinMOPAC 3.5Proを用いてモデルペプチド、N-Ac-Ser-Ile-Leu-Gln-Ala-Gly-X 1-X2-Glyの生成熟を算出した。X1、X2のアミノ酸配列は-SeCys-Cys-、-Cys-SeCys-、-SeCys-SeCys-、-Cys-Cys-のいずれかで、それぞれ酸化状態と還元状態を計算し、そのエンタルピー差を求めた。ハミルトニアンAM1とPM3は同じ傾向の計算結果を示し、セレノスルフィドまたはジセレニドを形成するペプチドは酸化状態でより安定化するのに対して-Cys-Cys-の配列を持つペプチドは還元形の方が安定であることを示した。ほ乳類のTrxRのSeCys498をCysに置換すると酵素活性が1％程度にまで低下することが報告されている。これは、変異酵素が-Cys497-Cys498-間で酸化的架橋を形成にくいからと考察されていたが、今回の分子軌道計算の結果は、この仮説を支持している。

好酸性細菌Acidocella facilis 22Mの制限修飾系遺伝子のクローニングと塩基配列決定

山岡誠司, 田村隆, 竹信尚典, 古城俊之, 田中英彦, 稲垣賢二

岡山大学農学部学術報告 92 9 - 15 2003年

記述言語：日本語出版者・発行元：岡山大學農學部

CGATCGを認識するAfa22MI制限修飾系遺伝子を好酸性細菌Acidocella facilis 22Mより、クローニングし、塩基配列を決定した。その結果、Ｃ５－シトシンメチラーゼに特徴的なモチーフが保存されたM.Afa22MI遺伝子、その上流に、M.Afa22MI遺伝子とは逆方向に、very short patch repair endonuclease様タンパク質遺伝子（Afa22MI vsr）、M.Afa22MI遺伝子の下流にM.Afa22MI遺伝子と同方向に、制限酵素（R.Afa22MI）遺伝子と推定されるオープンリーディングフレームが見出された。M.Afa22MIの推定アミノ酸配列は、Xanthomonas oryzae pv. oryzae由来M.XorIIの配列と全体で約63％、variable region内で約53％の高い配列類似性を示した。また、Afa22MI vsrの推定アミノ酸配列も、M.XorIIに付随するXorII vsrの配列と約66％の高い類似性を示した。

その他リンク： http://eprints.lib.okayama-u.ac.jp/10584

Semiemperical molecular orbital calculation for the redox property of C-terminal active site sequence of human thioredoxin reductase

T. Tamura, H. Tanaka, K. Inagaki

Sci. Rep. Fac. Agric. Okayama Univ 2003年

Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans

H Inoue, T Tamura, N Ehara, A Nishito, Y Nakayama, M Maekawa, K Imada, H Tanaka, K Inagaki

FEMS MICROBIOLOGY LETTERS 214 ( 1 ) 127 - 132 2002年8月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE BV

An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M-r 45 000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1097(02)00857-1

抗がん酵素L-Methionine γ-lyaseの精密立体構造

1.シネフンギン生合成酵素の遺伝子クローニング

田中英彦, 田村隆

ビタミン 76 ( 4 ) 241 - 241 2002年4月

山下真生, 田村隆, 瀧本明生, 光島健二, 江崎信芳, 左右田健次, 田中英彦, 稲垣賢二

ビタミン 76 ( 3 ) 177 - 177 2002年3月

An in vitro screening method for DNA cytosine-C-5-methylase inhibitor

T Tamura, A Kataoka, LY Shu, A Ashida, H Tanaka, K Inagaki

NATURAL PRODUCT LETTERS 16 ( 1 ) 25 - 27 2002年2月

記述言語：英語出版者・発行元：TAYLOR & FRANCIS LTD

A specific inhibitor of DNA cytosine C-5-methylases would be useful for studying genomic imprinting, X-chromosome inactivation, carcinogenesis, and regulation of tissue-specific gene expression, for these physiological phenomena appears to be regulated through DNA methylation in promoter sequences. This paper reports a novel convenient in vitro assay method for screening DNA cytosine C-5-Methylase inhibitor. Our method uses a commercially available Hae III methylase (cytosine C-5 methylase), its corresponding Hae III endonuclease, and lambda DNA as their substrate.

DOI： 10.1080/1057563029001/4809

好酸性細菌 Acidocella facilis 由来菌体外酸性エステラーゼ遺伝子のクローニング

Mammalian thioredoxin reductases

T Tamura, TC Stadtman

PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES, PT A, SELENOPROTEINS AND THIOREDOXIN 347 297 - 306 2002年

高橋竜也, 田中登, 田村隆, 向井千佳, 磯部公安, 若尾紀夫, 田中英彦, 稲垣賢二

岡山大学農学部学術報告 91 7 - 14 2002年

記述言語：日本語出版者・発行元：岡山大学

好酸性従属栄養細菌Acidocella facilis AIU409株は、基質としてソルビタンモノエステルであるTweenを含む培地中で培養された時に、菌体外に熱安定性の高い酸性エステラーゼを生産する。エステラーゼをコードする遺伝子(estA)をA. facilis AIU409株のゲノムライブラリーから単離し、大腸菌MV1184にクローニングし、その遺伝子の全塩基配列を決定した。その結果、estAの構造遺伝子が、1881塩基対であることが明らかになった。酸性エステラーゼ遺伝子のオープンリーディングフレーム(ORF)は、627アミノ酸残基（計算された分子量は64、140ダルトン）をコードしていた。ロー囚子非依存性の転写終結シグナルが終止コドンであるTGAのすぐ下流に存在していた。そのN末端予想アミノ酸配列より、酸性エステラーゼの前駆体は、N末端に23個のアミノ酸残基から成るシグナルペプチドを有していた。また、リパーゼのコンセンサス配列であるG-X-S-X-Sの配列が存在することが明らかとなっ。酸性エステラーゼの予想アミノ酸配列から計算された分子量は61,486であり、これはSDS-PAGEによって予想されていた分子量より、やや低い値であった。また、Acidocella facilis酸性エステラーゼの予想アミノ酸配列は、Aeromonas hydrophila由来のacyltrans-ferase12、13)やXenorhabdus luminescens由来のリパーゼ14)と高い相同性を示した。

Selenium for Cancer Prevention and Human Health

その他リンク： http://eprints.lib.okayama-u.ac.jp/10603

L-グルタミン酸オキシダーゼを用いたグルタミン酸センサーの開発及びGOT/GTPセンシングへの応用

有馬二朗, 田村隆, 篠原寛明, 日下部均, 田中英彦, 稲垣賢二

岡山大学農学部学術報告 2002年

Improvement of sinefungin-producing strain of Streptomyces incarnatus by conferring rifampicin-resistance through ultraviolet light irradiation and protoplast regeneration

Tamura, T, L.-Y. Shu, H. Tanaka, K. Inagaki

Sci. Rep. Fac. Agric. Okayama Univ. 2002年

Takashi Tamura, Hidehiko Tanaka, Kenji Inagaki

Foods and Foods Ingredients Journal of Japan 198, 24-38 ( 198 ) 24 - 38 2002年

2-III-15 ヒト肺がん細胞由来のチオレドキシン還元酵素遺伝子の点突然変異

田村隆, 長谷川雅哉, 稲垣賢二, 田中英彦

ビタミン 75 ( 4 ) 243 - 243 2001年4月

2-III-17 硫黄酸化細菌Thiobacillus thiooxidans由来イソクエン酸脱水素酵素と同遺伝子の解析

江原渚, 井上宏之, 田村隆, 田中英彦, 稲垣賢二

ビタミン 75 ( 4 ) 244 - 244 2001年4月

2-III-13 放線菌由来L-グルタミン酸オキシダーゼ : クローニング,発現,及び諸性質検討

有馬二朗, 崎川智代, 田村隆, 芦内誠, 八木年晴, 日下部均, 田中英彦, 稲垣賢二

ビタミン 75 ( 4 ) 242 - 243 2001年4月

2. Pseudomonas putida L-Methionine γ-lyaseのTyrosine114の役割

井上宏之, 稲垣賢二, 田村隆, 江崎信芳, 左右田健次, 田中英彦

ビタミン 75 ( 3 ) 154 - 155 2001年3月

Purification and characterization of an acid trehalase from Acidobacterium capsulatum

K Inagaki, N Ueno, T Tamura, H Tanaka

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 91 ( 2 ) 141 - 146 2001年2月

記述言語：英語出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

We purified an acid trehalase (EC 3.2.1.28, a,alpha,alpha'-trehalose glucohydrolase) from an acidophilic bacterium, Acidobacterium capsulatum. The enzyme was homogeneous based on polyacrylamide gel electrophoresis, and was composed of a single polypeptide chain with a molecular mass of 57 kDa. Maximum trehalase activity was observed at pH 2.5. The acid trehalase exhibited an apparent K-m of 1.0 mM for trehalose at 30 degreesC and pH 3.0, The trehalase was located in the periplasmic space. The activity of the enzyme was activated by 1.0 mM MnCl2 or CoCl2, and inhibited by 1.0 mM PbCl2, HgCl2, NiCl2, p-chloromercuribenzoate, N-ethylmaleimide, monoiodoacetate, or EDTA. The enzyme showed high specificity for trehalose. It was found that an equimolar mixture of alpha -D-glucose and beta -D-glucose was formed on hydrolysis of trehalose by the trehalase.

Molecular cloning of thioredoxin reductase isozymes, TrxR1 and TrxR2, from human lung adenocarcinoma cell line, NCI-H441

セレンは猛毒性・発癌性の栄養素?

田村隆

バイオアクティブ 11,15-18 2001年

Streptomyces sp X-119-6 由来L-グルタミン酸オキシダーゼのクローニング及び発現

有馬二朗, 崎川智代, 田村隆, 芦内誠, 八木年春, 日下部均, 田中英彦, 稲垣賢二

微量栄養素研究 2001年

Tamura, T, M. Hasegawa, M. Sugimoto, K. Inagaki, H. Tanaka

Biryo-eiyouso-kenkyu 18 149 - 153 2001年

709 放線菌由来L-グルタミン酸オキシダーゼを利用したL一GluセンサーとGOT・GPTセンシングへの応用

有馬二朗, 田村隆, 崎川智代, 金原陽平, 篠原寛明, 日下部均, 田中英彦, 稲垣賢二

日本生物工学会大会講演要旨集 13 289 - 289 2001年

116 19F-nmrを用いるモノフルオロ酢酸生産のCosynthesis実験

田村隆, 澤本有香子, 田中英彦, 稲垣賢二

日本生物工学会大会講演要旨集 13 106 - 106 2001年

Purification and some characteristics of a monomeric alanine racemase from an extreme thermophile, Thermus thermophilus

TK Seow, K Inagaki, T Nakamura, R Maeda, T Tamura, H Tanaka

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 90 ( 3 ) 344 - 346 2000年9月

記述言語：英語出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about 38,000. The enzyme was most active at pH 8 and 75 degreesC, and remained active after incubation at 80 degreesC for 30 min.

211 NAD^+:penicillamine ADP transferase高生産性Bacillus sphaericusの作成

A colourimetric screening method for microorganisms that have methionine γ-lyase activity

Lim, L.-C, T. Tamura, R. Rahim, M.Ali, H. Ghazali, K. Inagaki, H.Tanaka

Asia Pacific J.Mol. Biol. Biotech., 8 ( 1 ) 95 - 97 2000年6月

1-II-9 L-メチオニンγ-リアーゼの立体構造

稲垣賢二, 田村隆, 田中英彦, 本島浩之, 熊坂崇, 古市真木雄, 山本雅貴, 田中信夫, 江崎信芳, 左右田健次

ビタミン 74 ( 4 ) 197 - 197 2000年4月

出海卓也, 田村隆, 稲垣賢二, 左右田健次, 田中英彦

日本生物工学会大会講演要旨集 12 34 - 34 2000年

Purification and characterization of NAD:penicillamine ADP transferase from Bacillus sphaericus: A novel NAD-dependent enzyme catalyzing phosphoramide bond formation

Jun Yanagidani, Takashi Tamura, Kenji Inagaki, Kenji Soda, Hidehiko Tanaka

Journal of Biological Chemistry 274 ( 2 ) 795 - 800 1999年1月

記述言語：英語

A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (K(m) = 13.0 mM) and L-penicillamine (K(m) = 6.5 mM)
other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.

DOI： 10.1074/jbc.274.2.795

1. Thiobacillus thiooxidansのイソクエン酸脱水素酵素の性質と遺伝子クローニング

稲垣賢二, 井上宏之, 田村隆, 田中英彦

ビタミン 72 ( 10 ) 571 - 571 1998年10月

Gene Cloning and Characterization of an Acidic Xylanase from Acidobacteriu

Kenji Inagaki, Ken Nakahira, Kazuhisa Mukai, Takashi Tamura, Hidehiko Tanaka

Bioscience, Biotechnology and Biochemistry 62 ( 6 ) 1061 - 1067 1998年1月

記述言語：英語

The gene xynA encoding an acid endo-β-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65°C, and is stable pH between 3.0 and 8.0. The Km and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30°C are 3.5 mg/ml and 403 μmol/min/mg. © 1998 Taylor &amp
Francis Group, LLC.

DOI： 10.1271/bbb.62.1061

Alanine racemase from an acidophile, acidiphilium organovorum: Purification and characterization

Teck Keong Seow, Kenji Inagaki, Takashi Tamura, Kenji Soda, Hidehiko Tanaka

Bioscience, Biotechnology and Biochemistry 62 ( 2 ) 242 - 247 1998年

記述言語：英語

An alanine racemase (EC 5.1.1.1) from an acidophilic heterotrophic bacterium, Acidiphilium organovorum 13H, was purified and characterized. The enzyme had a dimeric structure with identical subunits of M, 33,000 each. Although A. organovorum 13H is an acidophile, the enzyme had its maximum velocity at pH 9, corresponding to its location in the cytoplasm. Activity was maximum between 50 and 60°C. For an enzyme from a mesophile, it was stable to heat, showing no loss of activity after a 30-min incubation at 65°C. The enzyme needed pyridoxal 5′. phosphate (PLP) as a cofactor for its activity, as seen from the loss of activity upon dialysis against PLP-free buffer containing hydroxylamine and its absorption maximum at 420 nm. Activity was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content studies found that 1 mole of enzyme contained 2 moles of PLP. The enzyme catalyzed the symmetric reversible racemization of alanine exclusively. © 1998, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.62.242

Pseudomonas putidaのL-メチオニン分解系オペロンの解析

井上宏之, 田村隆, 稲垣賢二, 田中英彦

岡山大学農学部学術報告 87 53 - 58 1998年

染料脱色微生物の検索, 単離及び, 脱色機構の解析

稲垣賢二, 川口将和, 田口隆章, 田村隆, 田中英彦

岡山大学農学部学術報告 87 47 - 51 1998年

Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry

Takashi Tamura, Yoshinori Araki, Seiji Yamaoka, Kenji Inagaki, Hidehiko Tanaka

Nucleic Acids Research 25 ( 20 ) 4162 - 4164 1997年10月

記述言語：英語掲載種別：書評論文，書評，文献紹介等

We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M.EcoRI, M.BamHI and M.HaeIII.

DOI： 10.1093/nar/25.20.4162

ゴ-ルドラッシュ時代を迎えたセレンの生化学研究 (特集最近の生命科学)

田村隆, 田中英彦

化学工業 48 ( 9 ) 720 - 726 1997年9月

5.ペニシラミンの微生物代謝の酵素化学 : NADの新しい生理機能 : ビタミンB研究委員会第357回会議研究発表要旨

左右田健次, 柳谷純, 田村隆, 稲垣賢二, 田中英彦

ビタミン 71 ( 7 ) 312 - 313 1997年7月

2-III-4 ADP-penicillamine合成酵素の精製と反応生成物の同定 : 第49回大会一般研究発表要旨

柳谷純, 田村隆, 稲垣賢二, 田中英彦, 左右田健次

ビタミン 71 ( 4 ) 210 - 210 1997年4月

1-II-7 Pseudomonas putidaのメチオニン誘導性の新規α-ケト酪酸脱水素酵素E1について : 第49回大会一般研究発表要旨

井上宏之, 西藤晃, 田村隆, 稲垣賢二, 田中英彦

ビタミン 71 ( 4 ) 179 - 179 1997年4月

476 Isolation and Characterisation of Alanine Racemase from Extreme Thermophile, Thermus thermophilus :

Keong Seow Teck, Inagaki Kenji, Nakamura Takeshi, Maeda Ritsuko, Tamura Takashi, Tanaka Hidehiko

日本生物工学会大会講演要旨集 9 164 - 164 1997年

Pseudomonas putidaのα-ケト酪酸デヒドロゲナーゼE1成分の大腸菌内での発現と諸性質

井上宏之, 江里口真一, 西藤晃, 田村隆, 稲垣賢二, 田中英彦

日本分子生物学会年会プログラム・講演要旨集 19 417 - 417 1996年8月

ヒト肺腫瘍細胞に高発現するセレン酵素, チオレドキシン還元酵素の諸性質

田村隆, 田中英彦, STADTMAN Thressa C.

日本分子生物学会年会プログラム・講演要旨集 19 558 - 558 1996年8月

Thiobacillus thiooxidansの3‐イソプロピルリンゴ酸脱水素酵素精製と性質

松波秀行, 川口洋, 中山ゆみ, 田村隆, 稲垣賢二, 田中英彦

日本分子生物学会年会プログラム・講演要旨集 19th 131 1996年7月

Thiobacillus thiooxidansのイソクエン酸脱水素酵素精製と性質

西藤晃, 松波秀行, 中山ゆみ, 川口洋, 田村隆, 稲垣賢二, 田中英彦

日本分子生物学会年会プログラム・講演要旨集 19th 131 1996年7月

Pseudomonas putidaのL-メチオニン分解系オペロンの転写調節因子の解析 : 微生物

井上宏之, 田村隆, 稲垣賢二, 江崎信芳, 左右田健次, 田中英彦

日本農藝化學會誌 70 104 - 104 1996年3月

Bacillus属由来ペニシラミン分解酵素の反応生成物の同定 : 酵素

柳谷純, 田村隆, 稲垣賢二, 田中英彦, 左右田健次

日本農藝化學會誌 70 156 - 156 1996年3月

新規セレン酵素,チオレドキシン還元酵素の精製と諸性質 : 動物

田村隆, 田中英彦, Stadtman Thressa C.

日本農藝化學會誌 70 205 - 205 1996年3月

A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity

Takashi Tamura, Thressa C. Stadtman

Proceedings of the National Academy of Sciences of the United States of America 93 ( 3 ) 1006 - 1011 1996年2月

記述言語：英語

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 μM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine
hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se- carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent K(m) values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 μM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

DOI： 10.1073/pnas.93.3.1006

A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity

Takashi Tamura, Thressa C. Stadtman

Proceedings of the National Academy of Sciences of the United States of America 93 ( 3 ) 1006 - 1011 1996年2月

記述言語：英語

DOI： 10.1073/pnas.93.3.1006

Antimicrobial activity against methicillin-resistant Staphylococcus aureus in the culture broth of Enterococcus faecalis TH10, an isolate from Malaysian fermentation food, Temph

大平猪一朗, 田村隆, 藤井望, 稲垣賢ニ, 田中英彦

Jpn. J. Dairy Food Sci. 45(4), 93-96 ( 4 ) A - 93-A-96 1996年

記述言語：日本語出版者・発行元：日本酪農科学会

Bioassay-directed screening for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcus faecalis TH 10, which was isolated from a Malaysian fermentation food, Temph. The anti-MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against various lactic acid bacteria, including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released peptide or protein which shows potent activity against species closely related to the bacteriocin-producing strain. E. faecalis has been reported to produce bacteriocin which has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH 10 strain did not mediate the lysis of rabbit and human erythrocytes.

DOI： 10.11465/milkscience.45.A-93

722 好酸性細菌Acidobacterium capsulatum由来のキシラナーゼの精製及び諸性質の検討

その他リンク： https://jlc.jst.go.jp/DN/JLC/20016775976?from=CiNii

中平健, 向井和久, 田村隆, 稲垣賢二, 田中英彦

日本生物工学会大会講演要旨集 8 211 - 211 1996年

Antimicrobial activity against methicillin-resistant Staphylococcus aureus in the culture broth of Enterococcus faecalis TH10, an isolate from Malaysian fermentation food, Temph.

大平猪一朗, 田村隆, 藤井望, 稲垣賢ニ, 田中英彦

Japanese Journal of Dairy and Food Science 45 ( 4 ) 93 - 93-A-96 1996年

記述言語：日本語出版者・発行元：日本酪農科学会

DOI： 10.11465/milkscience.45.A-93

<研究紹介>セレノシステイン含有ペプチドの合成と生化学的機能の研究

その他リンク： https://jlc.jst.go.jp/DN/JLC/20016775976?from=CiNii

微生物によるフッ素イオンの有機フッ素化合物への取り込み

江崎信芳, 田村隆, 和田大, 左右田健次

Biomedical research on trace elements 6 ( 3 ) 117 - 118 1995年12月

田村隆

岡山大学農学部学術報告 84 69 - 72 1995年2月

記述言語：日本語出版者・発行元：岡山大学

Selenium belongs to the Ⅵb group of the periodic table,and possesses both metallic and nonmetallic characteristics. Physicochemical properties of selenium resemble more or less those of sulfur,and selenium may be indiscriminately incorporated in place of sulfur in cellular constituents and disturb metabolism. Alkali disease and blind stagger disease of livestock are caused by selenium-polluted grass. Carcinogenic effect is also one of the marked biological properties of selenium. In spite of the toxic and carcinogenic effects,selenium is actually an essential trace element for bacteria,fish,and mammals. Life has exploied the high reactivity and unique characteristics of organoselenium compounds,especially in the form of selenoenzymes. Mammalian glutathione peroxidase(EC 1.11.1.9) has selenocysteine residue at the active site, and the enzyme plays a central role in the biological defense system against oxidative challenge by activated oxygens and radicals. The author has studied the low redox potential and high reactivity of selenium-containing compounds, and developed glutathione peroxidase mimics. Glutaselenone, a selenium analog of glutaheione, catalyzes a glutathione peroxidase like reaction in vitro. Studies on the mechanisum of glutaselenone-catalyzed reaction revealed that glutaselenone is converted to a selenosulfide conjugate with glutathione in its catalysis. Thioredoxins contain a conserved sequence,Cys-Gly-Pro-Cys, which is known to form intramolecular disulfide bond with consecutive β-turn conformations. THe peptide is expected to serve as an template for intramolecular selenosulfide bond formation when either the cysteine is replaced by selenocystein. A tetrapeptide,Secys-Gly-Pro-Cys, was synthesized chemically. It showed glutathione peroxidase-like activity three times as high as glutaselenone. The high catalytic activity is ascribed to an intramolecular selenosulfide bond formation in the catalytic reaction.セレンは2-、0、2+、4+、6+と多彩な酸化還元状態をとるという点で金属的な性質を持つ。また有機セレン化合物として求核的・求電子的な反応性に関与できるという点で酸素や硫黄などの典型元素のような振る舞いをする。生命は進化の過程で、セレンのユニークな化学的特性をうまく利用し、グルタチオンペルオキシダーゼなどのセレン酵素を獲得してきたと考えられる。セレン酵素の研究に触発されて、セレン酵素化合物を医薬品として利用する研究が進められており注目を集めている。このような背景のもと、グルタセレノンとテトラペプチドを合成した。とくにテトラペプチドはグルタセレノンの反応機構研究から生まれたペプチドである。今後さらに高い活性を持つ人工酵素がデザインされ、合成されるだろう。その時、鋳型として蛋白質や核酸、オリゴ糖などある特定のコンフォメーションをとる生体物質が利用されると思われる。

その他リンク： http://ousar.lib.okayama-u.ac.jp/11140

Synthesis of Fluoroacetate from Fluoride, Glyceral, and β-Hydroxypyruvate by Streptomyces cattleya

Journal of Bacteriology 177 ( 9 ) 2265 - 2269 1995年

The mutual sparing effects of selenium and vitamin E in animal nutrition may be further explained by the discovery that mammalian thioredoxin reductase is a selenoenzyme

T Tamura, Gladyshev, V, SY Liu, TC Stadtman

BIOFACTORS 5 ( 2 ) 99 - 102 1995年

記述言語：英語掲載種別：書評論文，書評，文献紹介等出版者・発行元：IOS PRESS

The recent discovery that mammalian thioredoxin reductase is a selenoprotein furnishes an additional explanation of the mutual sparing roles of selenium and vitamin E in cellular antioxidant systems. Thioredoxin reductases isolated from human lung adenocarcinoma cells, human Jurkat T-cells and HeLa cells contain selenocysteine which is located in a C- terminal tripeptide, -Cys-SeCys-Gly.

Synthesis of fluoroacetate from fluoride, glycerol, and β- hydroxypyruvate by Streptomyces cattleya

T. Tamura, M. Wada, N. Esaki, K. Soda

Journal of Bacteriology 177 ( 9 ) 2265 - 2269 1995年

記述言語：英語

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]β-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U- 14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C
about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]β-hydroxypyruvates to show that C-2 and C-3 of β-hydroxy pyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through β-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.

グルタチオンのセレンアナログ、グルタセレノンの触媒するグルタチオンペルオキシダーゼ反応の機構査読

江崎信芳, 田村隆, 中里仁, 左右田健次

Biomed. Res. Trace Elements,4,87-88 1993年

Synthesis and characterization of the selenium analog of glutathione disulfide

Takashi Tamura, Tadao Oikawa, Akira Ohtaka, Nobutaka Fujii, Nobuyoshi Esaki, Kenji Soda

Analytical Biochemistry 208 ( 1 ) 151 - 154 1993年

記述言語：英語

We synthesized the selenium analog of glutathione disulfide by a liquid phase method and named it glutaselenone (i.e., γ-L-glutamyl-L-selenocysteinylglycine) diselenide. The selenol of selenocysteine was protected by the p-methoxybenzyl group, which was removed by acidolysis with trifluoroacetic acid in the presence of thioanisol. The overall yield of the final product, glutaselenone diselenide, was about 9% based on the starting compound, Se-(p-methoxybenzyl)-L-selenocysteine. Glutaselenone diselenide showed a broad absorption band between 270 and 400 nm and circular dichroism bands around 270 nm (positive) and 330 nm (negative), which were attributable to diselenide bond. © 1993 Academic Press, Inc.

DOI： 10.1006/abio.1993.1021