研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    冨田 宏矢(トミタ ヒロヤ), トミタ ヒロヤ

所属(マスター)

  • 工学研究院 応用化学部門 生物工学分野

所属(マスター)

  • 工学研究院 応用化学部門 生物工学分野

researchmap

プロフィール情報

所属

  • 北海道大学, 大学院工学研究院, 助教

学位

  • 博士(工学)(京都大学)

プロフィール情報

  • 冨田
  • 宏矢
  • ID各種

    201801019088288310

所属

  • 北海道大学, 大学院工学研究院, 助教

業績リスト

研究分野

  • ナノテク・材料 / 生体化学
  • ナノテク・材料 / ケミカルバイオロジー
  • ライフサイエンス / 応用微生物学

経歴

  • 2021年04月 - 現在 北海道大学 大学院工学研究院 助教
  • 2020年04月 - 2021年03月 東京大学大学院 農学生命科学研究科 特任助教
  • 2017年04月 - 2020年03月 東京大学大学院 農学生命科学研究科 日本学術振興会特別研究員(PD)
  • 2015年02月 - 2017年03月 東京大学大学院 農学生命科学研究科 特任研究員
  • 2012年04月 - 2014年03月 京都大学大学院 工学研究科 日本学術振興会 特別研究員(DC2)

学歴

  • 2011年04月 - 2014年03月   京都大学大学院   工学研究科   合成・生物化学専攻 博士後期課程
  • 2009年04月 - 2011年03月   京都大学大学院   工学研究科   合成・生物化学専攻 修士課程
  • 2005年04月 - 2009年03月   京都大学   工学部   工業化学科

受賞

  • 2013年09月 International meeting on Thermophiles Best Poster Award
  • 2012年12月 極限環境生物学会 優秀ポスター賞

論文

  • Hiroya Tomita, Yohei Katsuyama, Yasuo Ohnishi
    Journal of industrial microbiology & biotechnology 2021年08月28日 [査読有り]
     
    Nitroaromatic compounds are essential materials for chemical industry, but they are also potentially toxic environmental pollutants. Therefore, their sensitive detection and degradation are important concerns. The microbial degradation pathways of nitroaromatic compounds have been studied in detail, but their usefulness needs to be evaluated to understand their potential applications in bioremediation. Here, we developed a rapid and relatively sensitive assay system to evaluate the activities and substrate specificities of nitroaromatic dioxygenases involved in the oxidative biodegradation of nitroaromatic compounds. In this system, nitrous acid, which was released from the nitroaromatic compounds by the dioxygenases, was detected and quantified using the Saltzman reagent. Escherichia coli producing the 3-nitrobenzoic acid dioxygenase complex MnbAB from Comamonas sp. JS46 clearly showed the apparent substrate specificity of MnbAB as follows. MnbAB accepted not only 3-nitrobenzoic acid but also several other p- and m-nitrobenzoic acid derivatives as substrates, although it much preferred 3-nitrobenzoic acid to others. Furthermore, the presence of a hydroxy or an amino group at the ortho position of the nitro group decreased the activity of MnbAB. In addition, MnbAB accepted 2-(4-nitrophenyl)acetic acid as a substrate, which has one additional methylene group between the aromatic ring and the carboxy group of 3-nitrobenzoic acid. This is the first report about the detailed substrate specificity of MnbAB. Our system can be used for other nitroaromatic dioxygenases and contribute to their characterization.
  • Seiji Kawai, Yuko Sugaya, Ryota Hagihara, Hiroya Tomita, Yohei Katsuyama, Yasuo Ohnishi
    Angewandte Chemie (International ed. in English) 2021年02月23日 [査読有り]
     
    DON (6-Diazo-5-oxo-L-norleucine), a diazo-containing amino acid, has been studied for more than 60 years as a potent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala-DON-DON which has antitumor activity, by gene inactivation and in vitro analysis of recombinant enzymes. We also established heterologous production of N-acetyl-DON in Streptomyces albus. DON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly, transmembrane protein AzpL was indicated to catalyze diazotization using 5-oxolysine and nitrous acid as substrates. Site-directed mutagenesis of AzpL indicated that the hydroxy group of Tyr-93 is important for the diazotization. These findings expand our knowledge of the enzymology of N-N bond formation.
  • Ren-Chao Zheng, Xia-Feng Lu, Hiroya Tomita, Shin-ichi Hachisuka, Yu-Guo Zheng, Haruyuki Atomi
    Journal of Bacteriology 2021年01月19日 [査読有り]
  • Eiichiro Kan, Hiroya Tomita, Yohei Katsuyama, Jun-Ichi Maruyama, Yasuji Koyama, Yasuo Ohnishi
    Chembiochem : a European journal of chemical biology 2020年09月03日 [査読有り]
     
    The filamentous fungus Aspergillus oryzae has 27 putative iterative type I polyketide synthase (PKS) gene clusters, but the secondary metabolites produced by them are mostly unknown. Here, we focused on eight clusters that were reported to be expressed at relatively high levels in a transcriptome analysis. By comparing metabolites between an octuple-deletion mutant of these eight PKS gene clusters and its parent strain, we found that A. oryzae produced 2,4'-dihydroxy-3'-methoxypropiophenone (1) and its precursor, 4'-hydroxy-3'-methoxypropiophenone (3) in a specific liquid medium. Furthermore, an iterative type I PKS (PpsB) encoded by AO090102000166 and an acetyl-CoA ligase (PpsA) encoded downstream from ppsB were shown to be essential for their biosynthesis. PpsC, encoded upstream from ppsB, was shown to have 3-binding activity (Kd =26.0±6.2 μM) and is suggested to be involved in the conversion of 3 to 1. This study deepens our understanding of cryptic secondary metabolism in A. oryzae.
  • Kita A, Kishimoto A, Shimosaka T, Tomita H, Yokooji Y, Imanaka T, Atomi H, Miki K
    Proteins 2019年11月 [査読有り][通常論文]
  • Zheng RC, Hachisuka SI, Tomita H, Imanaka T, Zheng YG, Nishiyama M, Atomi H
    The Journal of biological chemistry 293 10 3625 - 3636 2018年 [査読有り][通常論文]
     
    Aminotransferases are pyridoxal 5-phosphate– dependent enzymes that catalyze reversible transamination reactions between amino acids and -keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use L-ornithine (Orn), L-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis. We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high -aminotransferase activity with L-Orn and L-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene–disruption strain (△TK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of L-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.
  • Hiroya Tomita, Yohei Katsuyama, Hiromichi Minami, Yasuo Ohnishi
    JOURNAL OF BIOLOGICAL CHEMISTRY 292 38 15859 - 15869 2017年09月 [査読有り][通常論文]
     
    Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA-rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF. Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both in vitro and in vivo. Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of similar to 0.1 mu M. These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.
  • Takahiro Shimosaka, Hiroya Tomita, Haruyuki Atomi
    JOURNAL OF BACTERIOLOGY 198 14 1993 - 2000 2016年07月 [査読有り][通常論文]
     
    Regulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyper-thermophilic bacterium Thermotoga maritima harbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on the T. maritima genome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) from Corynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designated TmKPR/KARI. When T. maritima cell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting that Tm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity of Tm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties of Tm-PanK and the pantothenate synthetase (Tm-PS) of this organism. Tm-PS was not affected by CoA. Surprisingly however, Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 mu M CoA. Our results suggest that CoA biosynthesis in T. maritima is regulated by feedback inhibition targeting PanK, although Tm-PanK is a type III enzyme. IMPORTANCE Bacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II pantothenate kinase ( PanK). The hyperthermophilic bacterium Thermotoga maritima harbors a single type III PanK (Tm-PanK), previously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in this organism, we found that Tm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation of how CoA biosynthesis is regulated in T. maritima, which may also apply for other bacteria that harbor only type III PanK enzymes.
  • Yoshiki Aikawa, Yuichi Nishitani, Hiroya Tomita, Haruyuki Atomi, Kunio Miki
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 72 Pt 5 369 - 375 2016年05月 [査読有り][通常論文]
     
    Coenzyme A (CoA) plays pivotal roles in a variety of metabolic pathways in all organisms. The biosynthetic pathway of CoA is strictly regulated by feedback inhibition. In the hyperthermophilic archaeon Thermococcus kodakarensis, ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate, is a target of feedback inhibition by CoA. The crystal structure of KPR from T. kodakarensis (Tk-KPR) complexed with CoA and 2-oxopantoate has previously been reported. The structure provided an explanation for the competitive inhibition mechanism. Here, further biochemical analyses of Tk-KPR and the crystal structure of Tk-KPR in complex with NADP(+) are reported. A mutational analysis implies that the residues in the binding pocket cooperatively contribute to the recognition of CoA. The structure reveals the same dimer architecture as the Tk-KPR-CoA-2-oxopantoate complex. Moreover, the positions of the residues involved in the dimer interaction are not changed by the binding of CoA and 2-oxopantoate, suggesting individual conformational changes of Tk-KPR monomers.
  • Yoshiki Aikawa, Yuichi Nishitani, Hiroya Tomita, Haruyuki Atomi, Kunio Miki
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 84 3 374 - 382 2016年03月 [査読有り][通常論文]
     
    Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P) H-dependent reduction of 2-oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2-oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2-oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea.
  • Asako Kishimoto, Akiko Kita, Takuya Ishibashi, Hiroya Tomita, Yuusuke Yokooji, Tadayuki Imanaka, Haruyuki Atomi, Kunio Miki
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 82 9 1924 - 1936 2014年09月 [査読有り][通常論文]
     
    Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes to convert pantoate to 4 '-phosphopantothenate. These two enzymes are absent in almost all archea. Recently, it was reported that two novel enzymes, pantoate kinase, and phosphopantothenate synthetase (PPS), are responsible for this conversion in archea. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, thermococcus kodakarensis and its complexes with substrates, ATP, and ATP and 4-phosphopantoate. PPS forms an asymmetric homodinmer, in which two monomers composing a dimer, deviated from the exact twofold symmetry, displaying 4 degrees-13 degrees distortion. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on these structures, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate, which is thought to be a reaction intermediate.
  • Tomita H, Yokooji Y, Ishibashi T, Imanaka T, Atomi H
    Journal of bacteriology 196 6 1222 - 1230 2014年03月 [査読有り][通常論文]
     
    beta-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. beta-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of beta-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to gamma-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of beta-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the beta-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for beta-alanine biosynthesis in these organisms.
  • Tomotsugu Awano, Anja Wilming, Hiroya Tomita, Yuusuke Yokooji, Toshiaki Fukui, Tadayuki Imanaka, Haruyuki Atomi
    JOURNAL OF BACTERIOLOGY 196 1 140 - 147 2014年01月 [査読有り][通常論文]
     
    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative alpha subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two alpha subunits were each coexpressed with the beta subunit-encoding TK0943 gene. In both cases, soluble proteins with an alpha(2)beta(2) structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl) acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl) acetyl-CoA synthetase activity, and 2-(imidazol-4-yl) acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.
  • Hiroya Tomita, Tadayuki Imanaka, Haruyuki Atomi
    Molecular Microbiology 90 2 307 - 321 2013年10月 [査読有り][通常論文]
     
    Summary: Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20-29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk-KPR) displayed reductase activity specific for 2-oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes. Tk-KPR activity decreased dramatically in the presence of CoA and KPR activity in cell-free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T.kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea. © 2013 John Wiley & Sons Ltd.
  • Haruyuki Atomi, Hiroya Tomita, Takuya Ishibashi, Yuusuke Yokooji, Tadayuki Imanaka
    Biochemical Society Transactions 41 1 427 - 431 2013年02月 [査読有り][通常論文]
     
    CoA is a ubiquitous molecule in all three domains of life and is involved in various metabolic pathways. The enzymes and reactions involved in CoA biosynthesis in eukaryotes and bacteria have been identified. By contrast, the proteins/genes involved in CoA biosynthesis in archaea have not been fully clarified, and much has to be learned before we obtain a general understanding of how this molecule is synthesized. In the present paper, we review the current status of the research on CoA biosynthesis in the archaea, and discuss important questions that should be addressed in the near future. © 2013 Biochemical Society.
  • Takuya Ishibashi, Hiroya Tomita, Yuusuke Yokooji, Tatsuya Morikita, Bunta Watanabe, Jun Hiratake, Asako Kishimoto, Akiko Kita, Kunio Miki, Tadayuki Imanaka, Haruyuki Atomi
    EXTREMOPHILES 16 6 819 - 828 2012年11月 [査読有り][通常論文]
     
    We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4'-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and beta-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3 kJ mol(-1). In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with beta-alanine, but not with gamma-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis-Menten kinetics toward beta-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4'-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea.
  • Hiroya Tomita, Yuusuke Yokooji, Takuya Ishibashi, Tadayuki Imanaka, Haruyuki Atomi
    JOURNAL OF BACTERIOLOGY 194 19 5434 - 5443 2012年10月 [査読有り][通常論文]
     
    Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4'-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable k(cat)/K-m values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg2+, respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.
  • Sonoko Ishino, Seiji Fujino, Hiroya Tomita, Hiromi Ogino, Koichi Takao, Hiromi Daiyasu, Tamotsu Kanai, Haruyuki Atomi, Yoshizumi Ishino
    Genes to cells 16 12 1176 - 1189 2011年12月 [査読有り][通常論文]
  • Sonoko Ishino, Seiji Fujino, Hiroya Tomita, Hiromi Ogino, Koichi Takao, Hiromi Daiyasu, Tamotsu Kanai, Haruyuki Atomi, Yoshizumi Ishino
    GENES TO CELLS 16 12 1176 - 1189 2011年12月 [査読有り][通常論文]
     
    In eukaryotes, the replicative DNA helicase core is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2-7). Recent studies showed that the CMG (Cdc45MCMGINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog. It is thus, of high interest, whether and how these three Mcms share their functions in DNA metabolism in this hyperthermophile. Here, we report the biochemical properties of two of these proteins, TkoMcm1 and TkoMcm3. In addition, their physical and functional interactions with GINS, possibly an essential factor for the initiation and elongation process of DNA replication, are presented through in vitro ATPase and helicase assays, and an in vivo immunoprecipitation assay. Gene disruption and product quantification analyses suggested that TkoMcm3 is essential for cell growth and plays a key role as the main DNA helicase in DNA replication, whereas TkoMcm1 also shares some function in the cells.
  • Yuusuke Yokooji, Hiroya Tomita, Haruyuki Atomi, Tadayuki Imanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 41 28137 - 28145 2009年10月 [査読有り][通常論文]
     
    Bacteria/eukaryotes share a common pathway for coenzyme A (CoA) biosynthesis. Although archaeal genomes harbor homologs for most of these enzymes, homologs of bacterial/eukaryotic pantothenate synthetase (PS) and pantothenate kinase (PanK) are missing. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to produce pantothenate, whereas PanK catalyzes the ATP-dependent phosphorylation of pantothenate to produce 4'-phosphopantothenate. When we examined the cell-free extracts of the hyperthermophilic archaeon Thermococcus kodakaraensis, PanK activity could not be detected. A search for putative kinase-encoding genes widely distributed in Archaea, but not present in bacteria/eukaryotes, led to four candidate genes. Among these genes, TK2141 encoded a protein with relatively low PanK activity. However, higher levels of activity were observed when pantothenate was replaced with pantoate. V(max) values were 7-fold higher toward pantoate, indicating that TK2141 encoded a novel enzyme, pantoate kinase (PoK). A search for genes with a distribution similar to TK2141 led to the identification of TK1686. The protein product catalyzed the ATP-dependent conversion of phosphopantoate and beta-alanine to produce 4'-phosphopantothenate and did not exhibit PS activity, indicating that TK1686 also encoded a novel enzyme, phosphopantothenate synthetase (PPS). Although the classic PS/PanK system performs condensation with beta-alanine prior to phosphorylation, the PoK/PPS system performs condensation after phosphorylation of pantoate. Gene disruption of TK2141 and TK1686 led to CoA auxotrophy, indicating that both genes are necessary for CoA biosynthesis in T. kodakaraensis. Homologs of both genes are widely distributed among the Archaea, suggesting that the PoK/PPS system represents the pathway for 4'-phosphopantothenate biosynthesis in the Archaea.

MISC

  • 微生物のニトロ化戦略
    冨田 宏矢 生物工学会誌 バイオミディア 2019年12月
  • 冨田宏矢, 勝山陽平, 大西康夫 化学 73 (7) 70‐71 2018年07月01日 [査読無し][通常論文]
  • 超高熱性アーキアの補酵素A生合成経路に固有の酵素群の構造解析
    喜田昭子, 岸本麻子, 下坂天洋, 石橋拓也, 冨田宏矢, 横大路裕介, 今中忠行, 跡見晴幸, 三木邦夫 京都大学原子炉実験所第52回学術講演会 2018年01月 [査読無し][通常論文]
  • アーキアの補酵素A生合成経路に固有の酵素群の構造解析
    喜田昭子, 岸本麻子, 下坂天洋, 石橋拓也, 冨田宏矢, 横大路裕介, 今中忠行, 跡見晴幸, 三木邦夫 第17回日本蛋白質科学会年会 2017年06月 [査読無し][通常論文]
  • Crystallographic studies of two enzymes required for the distinct CoA production pathway in archaea.
    A. Kita, A. Kishimoto, T. Shimosaka, T. Ishibashi, H. Tomita, Y. Yokooji, T. Imanaka, H. Atomi, K. Miki Extremophiles 2016 2016年09月 [査読無し][通常論文]
  • 冨田宏矢, 横大路裕介, 跡見晴幸, 跡見晴幸 化学と生物 54 (2) 85‐93 2016年01月20日 [査読無し][通常論文]
  • Haruyuki Atomi, Hiroya Tomita, Yuusuke Yokooji, Takuya Ishibashi, Tadayuki Imanaka ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 248 2014年08月 [査読無し][通常論文]
  • Thermococcus kodakarensis由来ホスホパントテン酸合成酵素の基質複合体の結晶構造解析
    岸本麻子, 喜田昭子, 石橋拓也, 冨田宏矢, 横大路裕介, 跡見晴幸, 三木邦夫 第86回日本生化学会大会 2013年09月 [査読無し][通常論文]
  • 超好熱性アーキア由来ホスホパントテン酸合成酵素基質複合体のX線結晶構造解析
    岸本麻子, 喜田昭子, 石橋拓也, 冨田宏矢, 横大路祐介, 跡見晴幸, 三木邦夫 第13回日本蛋白質科学会年会 2013年06月 [査読無し][通常論文]
  • 超好熱性アーキア由来ホスホパントテン酸合成酵素の結晶構造解析
    岸本麻子, 喜田昭子, 石橋拓也, 冨田宏矢, 横大路祐介, 跡見晴幸, 三木邦夫 平成24年度日本結晶学会年会 2012年10月 [査読無し][通常論文]
  • 超好熱性アーキアのホスホパントテン酸合成酵素の結晶構造
    岸本麻子, 喜田昭子, 石橋拓也, 冨田宏矢, 横大路裕介, 跡見晴幸, 三木邦夫 第12回日本蛋白質科学会年会 2012年06月 [査読無し][通常論文]
  • 補酵素A生合成に関与するArchaea特有の新規酵素 phosphopantothenate synthetaseの酵素学的特性の解明
    石橋拓也, 冨田宏矢, 横大路裕介, 森北達弥, 渡辺文太, 平竹潤, 今中忠行, 跡見晴幸 第12回極限環境生物学会年会,長崎 2011年11月27日 [査読無し][通常論文]
  • アーキアにおける補酵素A生合成の鍵中間体4-phosphopantoic acidの化学合成
    森北達弥, 渡辺文太, 石橋拓也, 冨田宏矢, 横大路祐介, 今中忠行, 跡見晴幸, 平竹潤 第12回極限環境生物学会年会,長崎 2011年11月 [査読無し][通常論文]
  • 補酵素A生合成に関わるArchaea特有の新規酵素phosphopantothenate synthetaseの酵素学的解析
    石橋拓也, 冨田宏矢, 横大路裕介, 森北達弥, 渡辺文太, 平竹潤, 今中忠行, 跡見晴幸 日本Archaea研究会第24回講演会,鶴岡 2011年09月 [査読無し][通常論文]
  • 超好熱性アーキアThermococcus kodakaraensisの複製フォーク複合体解明に向けてのMCM,GINS複合体解析
    藤野誠司, 石野園子, 井田梨沙, 尾木野弘実, 冨田宏矢, 金井保, 跡見晴幸, 真柳浩太, 大山拓次, 森川耿右, 石野良純 生化学 ROMBUNNO.2P-0483 2010年 [査読無し][通常論文]

講演・口頭発表等

  • 有用ポリマー生産に向けた酵素工学的アプローチ  [招待講演]
    冨田 宏矢
    第一回 日本生物工学会北日本支部 若手シンポジウム 2021年08月

共同研究・競争的資金等の研究課題

  • 微生物を利用した新規生分解性材料の生産と分解の解析
    北海道ガス:大学研究支援
    研究期間 : 2021年04月 -2022年03月
  • 放線菌が持つ新たなニトロ化酵素の探索
    日本学術振興会:若手研究
    研究期間 : 2018年04月 -2020年03月 
    代表者 : 冨田 宏矢
  • 放線菌由来新規ニトロ化酵素の同定と芳香族ジアミン生産系の開発
    日本学術振興会:特別研究員奨励費
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 冨田 宏矢
  • アーキア特有の補酵素A生合成機構の全容解明
    日本学術振興会:特別研究員奨励費
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 冨田 宏矢


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