研究者詳細 - 田村　隆

論文 - 田村　隆

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Illumina-based transcriptomic analysis of the fast-growing leguminous tree Acacia crassicarpa: functional gene annotation and identification of novel SSR-markers

Shougo Ishio, Kazutaka Kusunoki, Michiko Nemoto, Tadayoshi Kanao, Takashi Tamura

Frontiers in Plant Science 15 2024年8月

担当区分：責任著者掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers Media SA

Acacia crassicarpa is a fast-growing leguminous tree that is widely cultivated in tropical areas such as Indonesia, Malaysia, Australia, and southern China. This tree has versatile utility in timber, furniture, and pulp production. Illumina sequencing of A. crassicarpa was conducted, and the raw data of 124,410,892 reads were filtered and assembled de novo into 93,317 unigenes, with a total of 84,411,793 bases. Blast2GO annotation, Benchmark Universal Single-Copy Ortholog evaluation, and GO-term classification produced a catalogue of unigenes for studying primary metabolism, phytohormone signaling, and transcription factors. Massive transcriptomic analysis has identified microsatellites composed of simple sequence repeat (SSR) loci representing di-, tri-, and tetranucleotide repeat units in the predicted open reading frames. Polymorphism was induced by PCR amplification of microsatellite loci located in several genes encoding auxin response factors and other transcription factors, which successfully distinguished 16 local trees of A. crassicarpa tested, representing potentially exploitable molecular markers for efficient tree breeding for plantation and biomass exploitation.

DOI： 10.3389/fpls.2024.1339958

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Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation. 国際誌

Koki Okada, Yu Morimoto, Yukine Shiraishi, Takashi Tamura, Shigeki Mayama, Takashi Kadono, Masao Adachi, Kentaro Ifuku, Michiko Nemoto

Marine biotechnology (New York, N.Y.) 2023年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 μg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.

DOI： 10.1007/s10126-023-10273-w

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Author Correction: Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota. 国際誌

Titouan Jaunet-Lahary, Tatsuro Shimamura, Masahiro Hayashi, Norimichi Nomura, Kouta Hirasawa, Tetsuya Shimizu, Masao Yamashita, Naotaka Tsutsumi, Yuta Suehiro, Keiichi Kojima, Yuki Sudo, Takashi Tamura, Hiroko Iwanari, Takao Hamakubo, So Iwata, Kei-Ichi Okazaki, Teruhisa Hirai, Atsuko Yamashita

Nature communications 14 ( 1 ) 6053 - 6053 2023年9月

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota. 国際誌

Nature communications 14 ( 1 ) 1730 - 1730 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

DOI： 10.1038/s41467-023-36883-5

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Recombinant expression using the tetrathionate hydrolase promoter in Acidithiobacillus ferrooxidans.

Tadayoshi Kanao, Tomoki Kunihisa, Shuji Ohgimoto, Megumi Ito, Chisa Murakami, Hisayuki Nakayama, Takashi Tamura, Kazuo Kamimura

Journal of bioscience and bioengineering 135 ( 3 ) 176 - 181 2023年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

In the iron- and sulfur-oxidizing acidophilic chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, tetrathionate hydrolase gene (Af-tth) is highly expressed during tetrathionate growth. The expression levels of Af-tth were specifically determined by quantitative reverse transcription-polymerase chain reaction and the expression ratios of S0/Fe2+ and S4O62-/Fe2+ were found to be 68 ± 21 and 181 ± 5, respectively. The transcriptional start site was identified by primer extension. Promoter regions of Af-tth were cloned into the expression shuttle vector pMPJC and GFP gene was under the direction of the regions. Green fluorescence was observed by UV irradiation in recombinant A. ferrooxidans harboring the plasmid colonies grown on tetrathionate. Furthermore, His-tagged Af-Tth was synthesized in the recombinant cells grown on tetrathionate. Recombinant, His-tagged Af-Tth in an active form, was rapidly purified through metal-affinity column chromatography, although recombinant Af-Tth was synthesized in the inclusion bodies of Escherichia coli and acid-refolding treatment was necessary to recover the activity. The specific activity of purified Af-Tth from recombinant A. ferrooxidans (2.2 ± 0.37 U mg-1) was similar to that of acid-refolded Af-Tth from recombinant E. coli (2.5 ± 0.18 U mg-1). This method can be applied not only to heterologous expression but also to homologous expression of target genes for modification or specific mutation in A. ferrooxidans cells.

DOI： 10.1016/j.jbiosc.2022.12.005

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Characterization and application of l-methionine γ-lyase Q349S mutant enzyme with an enhanced activity toward l-homocysteine

Atsushi Okawa, Haruhisa Handa, Eri Yasuda, Masaki Murota, Daizo Kudo, Takashi Tamura, Tomoo Shiba, Kenji Inagaki

Journal of Bioscience and Bioengineering 133 ( 3 ) 213 - 221 2022年3月

A new l-arginine oxidase engineered from l-glutamate oxidase

Yoshika Yano, Shinsaku Matsuo, Nanako Ito, Takashi Tamura, Hitoshi Kusakabe, Kenji Inagaki, Katsumi Imada

PROTEIN SCIENCE 30 ( 5 ) 1044 - 1055 2021年5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：WILEY

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 mu M. The linear relationship is maintained around 1 mu M of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

DOI： 10.1002/pro.4070

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Structural basis for substrate specificity of L-methionine decarboxylase

Atsushi Okawa, Tomoo Shiba, Masaya Hayashi, Yuki Onoue, Masaki Murota, Dan Sato, Junko Inagaki, Takashi Tamura, Shigeharu Harada, Kenji Inagaki

PROTEIN SCIENCE 30 ( 3 ) 663 - 677 2021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：WILEY

L-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B-6-dependent enzyme and catalyzes the non-oxidative decarboxylation of L-methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

DOI： 10.1002/pro.4027

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Correction to: Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. 国際誌

Michiko Nemoto, Sayako Iwaki, Hisao Moriya, Yuki Monden, Takashi Tamura, Kenji Inagaki, Shigeki Mayama, Kiori Obuse

Marine biotechnology (New York, N.Y.) 23 ( 1 ) 157 - 157 2021年2月

Multiple mutations in RNA polymerase β-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory. 国際誌

Saori Ogawa, Hitomi Shimidzu, Koji Fukuda, Naoki Tsunekawa, Toshiyuki Hirano, Fumitoshi Sato, Kei Yura, Tomohisa Hasunuma, Kozo Ochi, Michio Yamamoto, Wataru Sakamoto, Kentaro Hashimoto, Hiroyuki Ogata, Tadayoshi Kanao, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Bioscience, biotechnology, and biochemistry 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

DOI： 10.1093/bbb/zbab011

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Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride

Masaki Kitagawa, Nanako Ito, Yuya Matsumoto, Masaya Saito, Takashi Tamura, Hitoshi Kusakabe, Kenji Inagaki, Katsumi Imada

Journal of Structural Biology: X 5 100044 - 100044 2021年

Structural basis of strict substrate recognition of l ‐lysine α‐oxidase from Trichoderma viride

Hiroki Kondo, Masaki Kitagawa, Yuya Matsumoto, Masaya Saito, Marie Amano, Shigeru Sugiyama, Takashi Tamura, Hitoshi Kusakabe, Kenji Inagaki, Katsumi Imada

Protein Science 2020年9月

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. 査読国際誌

Michiko Nemoto, Sayako Iwaki, Hisao Moriya, Yuki Monden, Takashi Tamura, Kenji Inagaki, Shigeki Mayama, Kiori Obuse

Marine biotechnology (New York, N.Y.) 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

DOI： 10.1007/s10126-020-09976-1

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Novel method for L-methionine determination using L-methionine decarboxylase and application of the enzyme for L-homocysteine determination 査読国際誌

Atsushi Okawa, Masaya Hayashi, Junko Inagaki, Toshihide Okajima, Takashi Tamura, Kenji Inagaki

Bioscience, Biotechnology, and Biochemistry 0916-8451 (print) ( 1347-6947 (online) ) 1 - 9 2020年1月

Antiviral effect of sinefungin on in vitro growth of feline herpesvirus type 1. 査読

Yudai Kuroda, Haruka Yamagata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura, Ken Maeda

The Journal of antibiotics 72 ( 12 ) 981 - 985 2019年12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：Springer Science and Business Media {LLC}

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

DOI： 10.1038/s41429-019-0234-4

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量子化学が解き明かすピリドキサール酵素の反応多様性

田村隆

生化学 91 ( 2 ) 260 - 264 2019年4月

Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase. 査読国際誌

Shinya Sutoh, Yuko Uemura, Yuko Yamaguchi, Asako Kiyotou, Rena Sugihara, Makiko Nagayasu, Mihoko Kurokawa, Koreaki Ito, Naoki Tsunekawa, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

Biochimica et biophysica acta. Proteins and proteomics 1867 ( 3 ) 194 - 201 2019年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was -172 mV, representing a less-oxidizing catalysis than the wild type DsbA (-122 mV), but one that is closer to yeast protein disulfide isomerase (-175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of -172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

DOI： 10.1016/j.bbapap.2018.12.005

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Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri. 査読国際誌

Michiko Nemoto, Dongni Ren, Steven Herrera, Songqin Pan, Takashi Tamura, Kenji Inagaki, David Kisailus

Scientific reports 9 ( 1 ) 856 - 856 2019年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

DOI： 10.1038/s41598-018-37839-2

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An Internal Sequence Region Catalytically Essential for the Human Selenophosphate Synthetase 2 査読

Muneaki Takahata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

FREE RADICAL BIOLOGY AND MEDICINE 112 65 - 65 2017年11月

Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

Takashi Tamura, Naoki Tsunekawa, Michiko Nemoto, Kenji Inagaki, Toshiyuki Hirano, Fumitoshi Sato

SCIENTIFIC REPORTS 6 19742 2016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：NATURE PUBLISHING GROUP

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing delta-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

DOI： 10.1038/srep19742

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