Faculty Profiles - TAMURA Takashi

MISC - TAMURA Takashi

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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 2003.12

Language：English Publisher：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

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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 2003.9

Language：English Publisher：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

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

H Inoue, A Nishito, S Eriguchi, T Tamura, K Inagaki, H Tanaka

JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 23 ( 2-6 ) 265 - 271 2003.9

Language：English Publisher：ELSEVIER SCIENCE BV

alpha-Ketobutyrate decarboxylase encoded in the L-methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of M-r 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg2+ Mn2+, and Co2+. The enzyme showed high activity for alpha-ketobutyrate and alpha-keto-n-valerate rather than pyruvate, but the alpha-keto acids with increasing length of the side chain as well as branching, such as alpha-keto-n-caproate and alpha-keto-3-methylvalerate, were not used by the enzyme. The K-m values for alpha-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the k(cat)/K-m value(10.69 s(-1) mM(-1))for alpha-ketobutyrate was 29-fold greater than that for pyruvate. Thus, alpha-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of alpha-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00089-4

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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

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 2003.2

Language：Japanese Publisher：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。

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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

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

Language：Japanese Publisher：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。

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Other Link： http://eprints.lib.okayama-u.ac.jp/10584

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 2002.8

Language：English Publisher：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

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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 2002.4

抗がん酵素L-Methionine γ-lyaseの精密立体構造

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 2002.3

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 2002.2

Language：English Publisher：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

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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

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

Selenium for Cancer Prevention and Human Health

Foods & food ingredients journal of Japan 198, 24-38 ( 198 ) 24 - 38 2002

Mammalian thioredoxin reductases

T Tamura, TC Stadtman

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

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

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

Language：Japanese Publisher：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。

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Other Link： http://eprints.lib.okayama-u.ac.jp/10603

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 2001.4

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 2001.4

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 2001.4

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 2001.3

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 2001.2

Language：English Publisher：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.

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