Faculty Profiles - TAMURA Takashi

Papers - TAMURA Takashi

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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 2024.8

Authorship：Corresponding author Publishing type：Research paper (scientific journal) Publisher：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

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

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

Marine biotechnology (New York, N.Y.) 2023.12

Language：English Publishing type：Research paper (scientific journal)

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

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

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 2023.9

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

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 ) 1730 - 1730 2023.4

Language：English Publishing type：Research paper (scientific journal)

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

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 2023.3

Language：English Publishing type：Research paper (scientific journal)

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

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 2022.3

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 2021.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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

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 2021.2

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. International journal

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 2021.1

Language：English Publishing type：Research paper (scientific journal)

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

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

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 2020.9

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases. Reviewed International journal

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

Marine biotechnology (New York, N.Y.) 2020.6

Language：English Publishing type：Research paper (scientific journal)

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

Novel method for l-methionine determination using l-methionine decarboxylase and application of the enzyme for l-homocysteine determination. Reviewed International journal

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

Bioscience, biotechnology, and biochemistry 0916-8451 (print) ( 1347-6947 (online) ) 1 - 9 2020.1

Language：English Publishing type：Research paper (scientific journal)

For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10-200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC.Abbreviations: DTT: dithiothreitol; IPTG: isopropyl-β-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5'-phosphate.

DOI： 10.1080/09168451.2020.1715781

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

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

The Journal of antibiotics 72 ( 12 ) 981 - 985 2019.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 2019.4

Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase. Reviewed International journal

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 2019.3

Language：English Publishing type：Research paper (scientific journal)

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

Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri. Reviewed International journal

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

Scientific reports 9 ( 1 ) 856 - 856 2019.1

Language：English Publishing type：Research paper (scientific journal)

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

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

Muneaki Takahata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

FREE RADICAL BIOLOGY AND MEDICINE 112 65 - 65 2017.11

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 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Molecular cloning and characterization of L-methionine gamma-lyase from Streptomyces avermitilis Reviewed

Daizou Kudou, Eri Yasuda, Yoshiyuki Hirai, Takashi Tamura, Kenji Inagaki

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 120 ( 4 ) 380 - 383 2015.10

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

A pyridoxal 5'-phosphate-dependent methionine gamma-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to alpha-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.02.019

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

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

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 ( 7 ) 1130 - 1132 2015.7

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

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 2015.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Other Link： http://orcid.org/0000-0003-1342-8885

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

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

Genome Announcements 3 ( 4 ) e00715 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

File： Microbiology Resource Announcements-2015-Oshima-e00715-15.full.pdf

DOI： 10.1128/genomeA.00715-15

Sulfur Transport System for the formation of iron-sulfur cluster

Tamura Takashi, Asano Kaori

VITAMINS 89 ( 2 ) 80 - 82 2015

Mechanism of N_2 fixation and H_2 production by nitrogenase

Tamura Takashi

VITAMINS 89 ( 8 ) 409 - 412 2015

Glycerol kinase from Cellulomonas sp. NT3060 which has high resistance against preservatives Reviewed

Sogabe Atsushi, Kitabayashi Masao, Morishima Kenichi, Furukawa Miyoko, Hatta Takashi, Fukuda Yasuhisa, Nishise Hiroshi, Oka Masanori, Tamura Takashi, Inagaki Kenji

Seibutsu-kogaku Kaishi 92 ( 8 ) 402 - 409 2014.8

Language：Japanese Publisher：公益社団法人日本生物工学会

Glycerol kinase is suitable for clinical diagnostic use in levels of neutral fat. Glycerol kinase from Cellulomonas sp. NT3060 shows good stability against several preservatives such as N-methylisothiazolone and imidazolidinylurea. We have cloned glpK gene from Cellulomonas sp. NT3060. This glpK gene is composed of 1,518 bp nucleotides and encodes 505 amino acid residues. The glpK gene was expressed in Escherichia coli and Cellulomonas sp. glycerol kinase was purified to homogeneity by column chromatography. The glycerol kinase exhibited high tolerance to various preservatives compared to other glycerol kinases. This tolerance was found to be favorable feature as diagnostic reagent for quantity of triglyceride.

The Role of Amino Acid Residues in the Active Site of L-Methionine gamma-lyase from Pseudomonas putida Reviewed

Mitsuki Fukumoto, Daizou Kudou, Shouko Murano, Tomoo Shiba, Dan Sato, Takashi Tamura, Shigeharu Harada, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 ( 7 ) 1275 - 1284 2012.7

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine gamma-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel beta-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

DOI： 10.1271/bbb.110906

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

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 2012.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

2PT109 Structure of L-lysine α-oxidase from Trichoderma viride(The 50th Annual Meeting of the Biophysical Society of Japan)

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

Seibutsu Butsuri 52 S123 2012

Quantum Chemical Mechanism of Hydrogeanses

Tamura Takashi

VITAMINS 86 ( 7 ) 412 - 414 2012

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

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 2011.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 Reviewed

Mitsuhiko Kuwahara, Takashi Tamura, Kentaro Kawamura, Kenji Inagaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 ( 3 ) 516 - 521 2011.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Protein Involvement in Early Phase of Se Metabolism

Tamura Takashi, Okugochi Takahiro

VITAMINS 85 ( 7 ) 346 - 348 2011

Selenoproteins in response to ER stress

Tamura Takashi

VITAMINS 85 ( 1 ) 27 - 28 2011

9.高基質特異性L-アミノ酸オキシダーゼの基質認識機構の解析(第419回研究協議会研究発表要旨,ビタミンB研究委員会)

内海友宏, 有馬二朗, 田村隆, 稲垣賢二

ビタミン 84 ( 5 ) 274 - 275 2010

Selenoproteins as potential nutritional biomarkers for Se

Tamura Takashi

VITAMINS 84 ( 2 ) 77 - 79 2010

Identification and Purification of an L-Alanine Dehydrogenase Homolog that Catalyzes the Formation of L-Ornithine Lactam from Sinefungin-Producing Streptomyces incarnatus NRRL8089 Reviewed

FUKUDA Koji, TAMURA Takashi, SATO Kumi, INAGAKI Kenji

Actinomycetologica 24 ( 2 ) 63 - 65 2010

Thioredoxin Reductase 1 Is Important for Selenoprotein Biosynthesis in HeLa Cells Reviewed

Kurokawa S, Mihara H, Yokoyama I, Mochizuki M, Yodoi J, Tamura T, Kurihara T, Esaki N

Biomedical research on trace elements 19 ( 1 ) 84 - 87 2008.4

1.放線菌Streptomyces avermitilis由来L-メチオニンγ-リアーゼの精製と性質(第413回研究協議会研究発表要旨,ビタミンB研究委員会)

工藤大蔵, 田村隆, 稲垣賢二

ビタミン 82 ( 11 ) 617 - 617 2008

3.好熱好酸性古細菌Sulfolobus tokodaii由来シスタチオニンγ-シンターゼ(第410回研究協議会研究発表要旨,ビタミンB研究委員会)

柳谷昌彦, 田村隆, 稲垣賢二

ビタミン 82 ( 1 ) 61 - 61 2008

3. Cellulomonas属細菌由来のグリセロールキナーゼの特性と立体構造解析(第407回研究協議会研究発表要旨,ビタミンB研究委員会)

稲垣賢二, 田村隆, 曽我部敦

ビタミン 81 ( 5 ) 258 - 259 2007

8.抗腫瘍性B_6酵素L-メチオニンγ-リアーゼにおけるCys116の役割(第398回ビタミンB研究委員会研究発表要旨)

工藤大蔵, 田村隆, 稲垣賢二

ビタミン 79 ( 1 ) 43 - 44 2005

16.硫黄酸化細菌A.thiooxidansのNAD^+依存型イソクエン酸脱水素酵素(ビタミンB研究委員会第399回研究協議会研究発表要旨)

稲垣賢二, 田村隆

ビタミン 79 ( 3 ) 194 - 194 2005

1P036 Implication for reaction mechanism of bacterial NAD dependent isocitrate dehydrogenase based on crystal structure and quantum chemistry

Imada K., Tamura T., Takenaka R., Kobayashi I., Inagaki K., Namba K.

Seibutsu Butsuri 45 S40 2005

Identification of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic environments in Japan Reviewed

K Goto, Y Tanimoto, T Tamura, K Mochida, D Arai, M Asahara, M Suzuki, H Tanaka, K Inagaki

EXTREMOPHILES 6 ( 4 ) 333 - 340 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG TOKYO

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius-Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.

DOI： 10.1007/s00792-001-0262-3

Role of tyrosine 114 of L-methionine y-lyase from Pseudomonas putida

H Inoue, K Inagaki, N Adachi, T Tamura, N Esaki, K Soda, H Tanaka

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 64 ( 11 ) 2336 - 2343 2000.11

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

L-Methionine gamma -lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of gamma -family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine gamma -lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in k(cat) for alpha,gamma -elimination of L-methionine, while the K-m remained the same as the wild type enzyme. The Y114F mutant had the reduced k(cat) by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma -position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of k(cat) for alpha,beta -elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma -elimination process for L-methionine is the rate-limiting determination step in alpha,gamma -elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma -lyase is important in gamma -elimination of the substrate.

DOI： 10.1271/bbb.64.2336

Crystal structure of the pyridoxal 5 '-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida

H Motoshima, K Inagaki, T Kumasaka, M Furuichi, H Inoue, T Tamura, N Esaki, K Soda, N Tanaka, M Yamamoto, H Tanaka

JOURNAL OF BIOCHEMISTRY 128 ( 3 ) 349 - 354 2000.9

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 Angstrom resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.

DOI： 10.1093/oxfordjournals.jbchem.a022760

Noncompetitive, reversible inhibition of aminoacylase-1 by a series of L-alpha-hydroxyl and L-alpha-fluoro fatty acids: Ligand specificity of Aspergillus oryzae and porcine kidney enzymes

T Tamura, Y Oki, A Yoshida, T Kuriyama, H Kawakami, H Inoue, K Inagaki, H Tanaka

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 379 ( 2 ) 261 - 266 2000.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

L-Lactate and L-beta-phenyllactate have been identified in the culture broth of Streptomyces sp. KY-11 as reversible noncompetitive inhibitors of Aspergillus oryzae aminoacylase-l and porcine kidney aminoacylase I. A series of alpha-hydroxyl acids (DL-R-CH(OH)-COOH, R = Et, n-pro, n-butyl, n-pentyl, n-hexyl) also inhibited the two enzymes in reversible noncompetitive kinetics, and the inhibition potency (-log K-i) increased with the increased hydrophobicity of the R group. The two eukaryotic enzymes showed distinct preferences to the ligand alpha-alkyl group, and the fungus enzyme was inhibited by L-beta-phenyllactate (R = benzyl) 10(3)-fold more potently than the mammalian enzyme. L-alpha-Fluoro-beta-phenyl-propionate and its D-isomer were used to show that the L-configuration of the alpha-substituent was important for potent inhibition of both the enzymes. The fungus aminoacylase-1 steeply decreased the affinity to alpha-fluoro-and alpha-hydroxy-n-caproate as pH was raised from 7 to 11, whereas the mammalian enzyme retained the affinity to these ligands under alkaline conditions. These results suggest that A. oryzae aminoacylase-1 has an acidic residue that interacts with OH or -F, while the mammalian enzyme would have a basic residue that recognizes the alpha-substituents. (C) 2000 Academic Press.

DOI： 10.1006/abbi.2000.1869

Purification of Anti-Escherichia Coli O-157 components produced by Enterococcus faecalis THIO, an isolate from Malaysian fermentation food, tempeh

Ohhira, I, T. Tamura, H. Kurokawa, H. Nakaue, K. Inagaki, H. Tanaka

Milk Science 49 ( 2 ) 81 - 88 2000

Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida Reviewed

H Inoue, K Inagaki, SI Eriguchi, T Tamura, N Esaki, K Soda, H Tanaka

JOURNAL OF BACTERIOLOGY 179 ( 12 ) 3956 - 3962 1997.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine gamma-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and fi, Tanaka, J. Biochem, (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex, A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of alpha-keto acid dehydrogenase complex were not found, When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for alpha-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important role in the metabolism of alpha-ketobutyrate produced by MdeA from L-methionine, Accordingly, MdeB encodes a novel E1 component, alpha-ketobutyrate dehydrogenase E1 component, of an unknown alpha-keto acid dehydrogenase complex in P. putida, In addition, we found that the mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucine-responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.

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