研究者詳細 - 田村　隆

論文 - 田村　隆

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Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis. 査読

Kudou D, Yasuda E, Hirai Y, Tamura T, Inagaki K

Journal of bioscience and bioengineering 120 ( 4 ) 380 - 383 2015年10月

Reactivity of sorbose dehydrogenase from Sinorhizobium sp 97507 for 1,5-anhydro-D-glucitol 査読

Toshio Araki, Tomoko Nakatsuka, Fuminao Kobayashi, Emi Watanabe-Ishimaru, Hirokazu Sanada, Takashi Tamura, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 ( 7 ) 1130 - 1132 2015年7月

Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, l-lysine alpha-oxidase from Trichoderma viride* 査読

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

JOURNAL OF BIOCHEMISTRY 157 ( 6 ) 549 - 559 2015年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

L-Lysine alpha-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form alpha-keto-epsilon-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 <remove> resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

DOI： 10.1093/jb/mvv012

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その他リンク： http://orcid.org/0000-0003-1342-8885

Draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin 査読

Kenshiro Oshima, Masahira Hattori, Hitomi Shimizu, Koji Fukuda, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Genome Announcements 3 ( 4 ) e00715 2015年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.

添付ファイル： Microbiology Resource Announcements-2015-Oshima-e00715-15.full.pdf

DOI： 10.1128/genomeA.00715-15

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鉄硫黄クラスター形成に関わる硫黄輸送システム

田村隆, 浅野香織

ビタミン 89 ( 2 ) 80 - 82 2015年

ニトロゲナーゼの窒素固定と水素生産の電子機構

田村隆

ビタミン 89 ( 8 ) 409 - 412 2015年

セルロモナスNT3060株の防腐剤耐性グリセロールキナーゼの遺伝子クローニングと特性解析査読

曽我部敦, 北林雅夫, 森島賢一, 古川美代子, 八田貴, 福田靖久, 西瀬弘, 岡正則, 田村隆, 稲垣賢二

生物工学会誌 92 ( 8 ) 402 - 409 2014年8月

記述言語：日本語出版者・発行元：公益社団法人日本生物工学会

グリセロールキナーゼは,中性脂肪測定用臨床検査薬用原料として利用されている.臨床検査薬は,近年溶液状態の検査薬が主流になり,長期間の保存を可能にするため,防腐剤を添加する事が一般的である.しかし,これらの防腐剤は酵素の安定性へ悪影響を及ぼす事もあり,臨床検査薬用原料酵素には溶液中の安定性に加え,これらの防腐剤への耐性も求められるようになっている.我々はCellulomonas sp. NT3060由来グリセロールキナーゼがN-methylisothiazoloneやimidazolidinylureaのような防腐剤に対し優れた安定性を示す事を見いだし,その酵素学的性質を明らかにした.Cellulomonas sp. NT3060由来のグリセロールキナーゼは,1,518塩基にコードされ,505アミノ酸より構成されていた.さらに,本遺伝子を導入した大腸菌により発現されたCellulomonas sp. NT3060由来グリセロールキナーゼを各種カラムクロマトグラフィーにより均一に精製し,その酵素学的特性を明らかにした.本グリセロールキナーゼの安定性は好熱性細菌由来のグリセロールキナーゼと比較すると不安定であるが,37℃付近の実用温度では十分安定であり,弱酸性領域から弱アルカリ領域まで幅広いpH領域で安定であった.また,基質であるグリセロールに対してKm値は6.9μMと高い親和性を示した.さらに,他起源のグリセロールキナーゼと比較して複数の防腐剤に対して高い耐性を示した.以上の性質より,本グリセロールキナーゼは臨床診断薬用原料として総合的に優れた性質を有した酵素である事が示唆された.

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The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida. 査読

Fukumoto M, Kudou D, Murano S, Shiba T, Sato D, Tamura T, Harada S, Inagaki K

Bioscience, biotechnology, and biochemistry 76 ( 7 ) 1275 - 1284 2012年7月

Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition 査読

Tomohiro Utsumi, Jiro Arima, Chika Sakaguchi, Takashi Tamura, Chiduko Sasaki, Hitoshi Kusakabe, Shigetoshi Sugio, Kenji Inagaki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 417 ( 3 ) 951 - 955 2012年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using.-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.12.033

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2PT109 L-リシン酸化酵素のX線結晶構造解析(日本生物物理学会第50回年会(2012年度))

Sano Tadahisa, Kawaguchi Tatsuya, Amano Marie, Shinyashiki Kengo, Nakata Haruka, Tamura Takashi, Inagaki Kenji, Kusakabe Hitoshi, Imada Katsumi

生物物理 52 S123 2012年

量子化学が解き明かすヒドロゲナーゼの触媒機構

田村隆

ビタミン 86 ( 7 ) 412 - 414 2012年

Selenite Reduction by the Thioredoxin System: Kinetics and Identification of Protein-Bound Selenide 査読

Takashi Tamura, Kumi Sato, Kentaro Komori, Takeshi Imai, Mitsuhiko Kuwahara, Takahiro Okugochi, Hisaaki Mihara, Nobuyoshi Esaxi, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 ( 6 ) 1184 - 1187 2011年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：TAYLOR & FRANCIS LTD

Selenite (SeO32-) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/auto-radiography using Se-75-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).

DOI： 10.1271/bbb.100847

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Identification and Conformer Analysis of a Novel Redox-Active Motif, Pro-Ala-Ser-Cys-Cys-Ser, in Drosophila Thioredoxin Reductase by Semiempirical Molecular Orbital Calculation 査読

Mitsuhiko Kuwahara, Takashi Tamura, Kentaro Kawamura, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 ( 3 ) 516 - 521 2011年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：TAYLOR & FRANCIS LTD

Mammalian thioredoxin reductases (TrxRs) contain selenium as selenocysteine (Sec) in the C-terminal redox center -Gly-Cys-Sec-Gly-OH to reduce Trx and other substrates; a Sec-to-Cys substitution in mammalian TrxR yields an almost inactive enzyme. The corresponding tetrapeptide sequence in Drosophila melanogaster TrxR (Dm-TrxR), -Ser-Cys-Cys-Ser-OH, endows the orthologous enzyme with a catalytic competence similar to mammalian selenoenzymes, but implementation of the Ser-containing tetrapeptide sequence SCCS into the mammalian enzyme does not restore the activity of the Sec-to-Cys mutant form (turnover number < 2/min). MOPAC calculation suggested that the C-terminal hexapeptide Pro-Ala-Ser-Cys-Cys-Ser-OH functions as a redox center that alleviates the necessity for selenium in Dm-TrxR, and a mutant form of human lung TrxR that mimics this hexapeptide sequence showed improved catalytic turnover (17.4/min for DTNB and 13.2/min for E. coli trx) compared to the Sec-to-Cys mutant. MOPAC calculation also suggested that the dominant form of the Pro-containing hexapeptide is a C+ conformation, which perhaps has a catalytic advantage in facile reduction of the intramolecular disulfide bond between Cys497 and Cys498 by the N-terminal redox center in the neighboring subunit.

DOI： 10.1271/bbb.100740

田村隆, 奥河内貴大

ビタミン 85 ( 7 ) 346 - 348 2011年