Faculty Profiles - TAMURA Takashi

MISC - TAMURA Takashi

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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 2008.7

Language：English Publisher：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

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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 2008.6

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)

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 2008.4

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 2008.4

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 2008.4

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 2008.4

Language：English Publisher：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

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Purification, characterization and crystal structure of isoamylase from thermophilic bacteria Rhodothermus marinus

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 2008.2

Language：Japanese Publisher：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．

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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 2008.1

Language：English Publisher：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

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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

2Fa07 Antitumor enzyme L-Lysine α-oxidase from filamentous fungi

NAKATA Haruka, TAMURA Takashi, INAGAKI Junko, KUSAKABE Hitoshi, INAGAKI Kenji

日本生物工学会大会講演要旨集 20 159 - 159 2008

1Fp11 Thermostabilization of glycerol kinase by ancestral mutation

Fukuda Yasuhisa, Tmura Takashi, Abe Asuka, Kishimoto Takahide, Sogabe Atsushi, Inagaki Kenji

20 155 - 155 2008

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 2007.8

Language：English Publisher：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

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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 2007.4

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 2007.4

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 2007.4

Structure of the antitumour enzyme L-methionine gamma-lyase from pseudomonas putida at 1.8 angstrom resolution

Daizou Kudou, Shintaro Misaki, Masao Yamashita, Takashi Tamura, Tomoaki Takakura, Takayuki Yoshioka, Shigeo Yagi, Robert M. Hoffman, Akio Takimoto, Nobuyoshi Esaki, Kenji Inagaki

JOURNAL OF BIOCHEMISTRY 141 ( 4 ) 535 - 544 2007.4

Language：English Publisher：JAPANESE BIOCHEMICAL SOC

L-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multi-functional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 angstrom resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.

DOI： 10.1093/jb/mvm055

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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 2007.2

Language：Japanese Publisher：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。

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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

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

1C11-4 Relationship between the structure of L-Glutamate oxidase and its enzymological characteristics

ARIMA Jiro, SASAKI Chiduko, SAKAGUCHI Chika, TAMURA Takashi, KUSAKABE Hitoshi, SUGIO Shigetoshi, INAGAKI Kenji

19 79 - 79 2007

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