Faculty Profiles - TAMURA Takashi

MISC - TAMURA Takashi

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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)

桑原光彦, 田村隆, 稲垣賢二

ビタミン 84 ( 4 ) 214 - 214 2010.4

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 2010.4

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 2010.4

Identification of Ornithine-lactam Converted from Arginine in Streptomyces incarnatus NRRL8089

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 2010.2

Language：Japanese Publisher：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．

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Purification and Characterization of Cystathionine γ-Synthase from Thermoacidophilic Archaea Sulfolobus tokodaii

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 2010.2

Language：Japanese Publisher：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．

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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

3P-1075 Purification and crystallization of antitumor enzyme, an L-lysine α-oxidase from Trichoderma viride

SHINYASHIKI Kengo, NAKATA Haruka, IMADA Katsumi, TAMURA Takashi, INAGAKI Kenji

22 76 - 76 2010

2P-1053 Expression of E. colt DsbA[CRIC] mutant has redox potential of Disulfide Isomerase

KIYOTOU Asako, TAMURA Takashi, YOSHIDA Takamasa, INAGAKI Kenji

22 22 - 22 2010

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 2009.7

Language：English Publisher：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

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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 2009.4

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 2009.4

Expression, Purification and Properties of Alanine Racemase from Thermus thermophillus HB8

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 2009.2

Language：Japanese Publisher：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。

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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 2009.1

Language：English Publisher：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

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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