研究者総覧

渡邉 誠也 (ワタナベ セイヤ)

  • 大学院農学研究科 生命機能学専攻 教授
Last Updated :2020/11/10

研究者情報

学位

  • 理学博士(北海道大学)

J-Global ID

研究キーワード

  • 鉄硫黄クラスター   アコニターゼ   糖代謝経路   遺伝子クラスター   微生物酵素   

研究分野

  • ライフサイエンス / 機能生物化学

経歴

  • 2016年04月 - 現在  愛媛大学大学院農学研究科Graduate School of Agriculture教授
  • 2010年12月 - 2016年03月  愛媛大学農学部Faculty of Agriculture准教授
  • 2010年05月 - 2010年11月  京都大学大学大学院農学研究科研究員
  • 2007年10月 - 2010年04月  京都大学大学エネルギー理工学研究所特任助教
  • 2005年04月 - 2007年09月  京都大学大学院工学研究科Graduate School of Engineering研究員
  • 2003年04月 - 2005年03月  京都大学国際融合創造センター研究員

学歴

  •         - 2003年   北海道大学   理学研究科   生物科学専攻(生物学)
  •         - 2003年   北海道大学   Graduate School, Division of Natural Science
  •         - 2000年   北海道大学   理学研究科   生物科学専攻(生物学)
  •         - 2000年   北海道大学   Graduate School, Division of Natural Science

所属学協会

  • 日本農芸化学会   日本生化学会   日本生物工学会   

研究活動情報

論文

  • Kentaroh Yoshiwara, Seiya Watanabe, Yasunori Watanabe
    Biochemical and biophysical research communications 530 1 203 - 208 2020年09月 [査読有り]
     研究論文(学術雑誌) 
    L-Arabinose 1-dehydrogenase (AraDH) is responsible for the first step of the non-phosphorylative L-arabinose pathway from bacteria, and catalyzes the NAD(P)+-dependent oxidation of L-arabinose to L-arabinonolactone. This enzyme belongs to the so-called Gfo/Idh/MocA protein superfamily, but has a very poor phylogenetic relationship with other functional members. We previously reported the crystal structures of AraDH without a ligand and in complex with NADP+. To clarify the underlying catalytic mechanisms in more detail, we herein elucidated the crystal structure in complex with L-arabinose and NADP+. In addition to the previously reported five amino acid residues (Lys91, Glu147, His153, Asp169, and Asn173), His119, Trp152, and Trp231 interacted with L-arabinose, which were not found in substrate recognition by other Gfo/Idh/MocA members. Structure-based site-directed mutagenic analyses suggested that Asn173 plays an important role in catalysis, whereas Trp152, Trp231, and His119 contribute to substrate binding. The preference of NADP+ over NAD+ was significantly subjected by a pair of Ser37 and Arg38, whose manners were similar to other Gfo/Idh/MocA members.
  • Seiya Watanabe, Yasunori Watanabe, Rika Nobuchi, Akari Ono
    Biochemistry 59 32 2962 - 2973 2020年08月 [査読有り]
     研究論文(学術雑誌) 
    l-2-Keto-3-deoxyarabinonate (l-KDA) dehydratase (AraD) catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde in the nonphosphorylative l-arabinose pathway from bacteria and belongs to the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein superfamily. All members of this superfamily, including several aldolases for l-KDA, share a common catalytic mechanism of retro-aldol fission, in which a lysine residue forms a Schiff base with the carbonyl C2 atom of the substrate, followed by proton abstraction of the substrate by a tyrosine residue as the base catalyst. Only AraD possesses a glutamine residue instead of this active site tyrosine, suggesting its involvement in catalysis. We herein determined the crystal structures of AraD from the nitrogen-fixing bacterium Azospirillum brasilense and AraD in complex with β-hydroxypyruvate and 2-oxobutyrate, two substrate analogues, at resolutions of 1.9, 1.6, and 2.2 Å, respectively. In both of the complexed structures, the ε-nitrogen of the conserved Lys171 was covalently linked to the carbonyl C2 atom of the ligand, which was consistent with the Schiff base intermediate form, similar to other DHDPS/NAL members. A site-directed mutagenic study revealed that Glu173 and Glu200 played important roles as base catalysts, whereas Gln143 was not absolutely essential. The abstraction of one of the C3 protons of the substrate (but not the O4 hydroxyl) by Glu173 was similar to that by the (conserved) tyrosine residues in the two DHDPS/NAL members that produce α-ketoglutaric semialdehyde (d-5-keto-4-deoxygalactarate dehydratase and Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase), indicating that these enzymes evolved convergently despite similarities in the overall reaction.
  • Yasunori Watanabe, Seiya Watanabe, Yasutaka Fukui, Hisashi Nishiwaki
    Biochemical and biophysical research communications 528 1 21 - 27 2020年07月 [査読有り]
     研究論文(学術雑誌) 
    Mutarotases catalyze the α-β anomeric conversion of monosaccharide, and play a key role in utilizing sugar as enzymes involved in sugar metabolism have specificity for the α- or β-anomer. In spite of the sequential similarity to l-rhamnose mutarotase protein superfamily (COG3254: RhaM), the ACAV_RS08160 gene in Acidovorax avenae ATCC 19860 (AaFucM) is located in a gene cluster related to non-phosphorylative l-fucose and l-galactose metabolism, and transcriptionally induced by these carbon sources; therefore, the physiological role remains unclear. Here, we report that AaFucM possesses mutarotation activity only toward l-fucose by saturation difference (SD) NMR experiments. Moreover, we determined the crystal structures of AaFucM in the apo form and in the l-fucose-bound form at resolutions of 2.21 and 1.75 Å, respectively. The overall structural folding was clearly similar to the RhaM members, differed from the known l-fucose mutarotase (COG4154: FucU), strongly indicating their convergent evolution. The structure-based mutational analyses suggest that Tyr18 is important for catalytic action, and that Gln87 and Trp99 are involved in the l-fucose-specific recognition.
  • Yasunori Watanabe, Yasuo Watanabe, Seiya Watanabe
    Structure (London, England : 1993) 28 7 799 - 809 2020年07月 [査読有り]
     研究論文(学術雑誌) 
    In both prokaryotes and eukaryotes, phosphatidylethanolamine (PE), one of the most abundant membrane phospholipids, plays important roles in various membrane functions and is synthesized through the decarboxylation of phosphatidylserine (PS) by PS decarboxylases (PSDs). However, the catalysis and substrate recognition mechanisms of PSDs remain unclear. In this study, we focused on the PSD from Escherichia coli (EcPsd) and determined the crystal structures of EcPsd in the apo form and PE-bound form at resolutions of 2.6 and 3.6 Å, respectively. EcPsd forms a homodimer, and each protomer has a positively charged substrate binding pocket at the active site. Structure-based mutational analyses revealed that conserved residues in the pocket are involved in PS decarboxylation. EcPsd has an N-terminal hydrophobic helical region that is important for membrane binding, thereby achieving efficient PS recognition. These results provide a structural basis for understanding the mechanism of PE biosynthesis by PSDs.
  • Yasunori Watanabe, Yasushi Tamura, Chika Kakuta, Seiya Watanabe, Toshiya Endo
    The Journal of biological chemistry 295 10 3257 - 3268 2020年03月 [査読有り]
     研究論文(学術雑誌) 
    Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of human VAT-1, a cytosolic soluble protein that was suggested to transfer phosphatidylserine, at 2.2 Å resolution. We found that VAT-1 transferred not only phosphatidylserine but also other acidic phospholipids between membranes in vitro Structure-based mutational analyses showed the presence of a possible lipid-binding cavity at the interface between the two subdomains, and two tyrosine residues in the flexible loops facilitated phospholipid transfer, likely by functioning as a gate to this lipid-binding cavity. We also found that a basic and hydrophobic loop with two tryptophan residues protruded from the molecule and facilitated binding to the acidic-lipid membranes, thereby achieving efficient phospholipid transfer.
  • Seiya Watanabe
    The Journal of biological chemistry 295 5 1338 - 1349 2020年01月 [査読有り]
     研究論文(学術雑誌) 
    The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown "route I" nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.
  • Seiya Watanabe, Fumiyasu Fukumori, Yasuo Watanabe
    Molecular microbiology 112 1 147 - 165 2019年07月 [査読有り]
     研究論文(学術雑誌) 
    The gene context in microorganism genomes is of considerable help for identifying potential substrates. The C785_RS13685 gene in Herbaspirillum huttiense IAM 15032 is a member of the d-altronate dehydratase protein family, and which functions as a d-arabinonate dehydratase in vitro, is clustered with genes related to putative pentose metabolism. In the present study, further biochemical characterization and gene expression analyses revealed that l-xylonate is a physiological substrate that is ultimately converted to α-ketoglutarate via so-called Route II of a non-phosphorylative pathway. Several hexonates, including d-altronate, d-idonate and l-gluconate, which are also substrates of C785_RS13685, also significantly up-regulated the gene cluster containing C785_RS13685, suggesting a possibility that pyruvate and d- or l-glycerate were ultimately produced (novel Route III). On the contrary, ACAV_RS08155 of Acidovorax avenae ATCC 19860, a homologous gene to C785_RS13685, functioned as a d-altronate dehydratase in a novel l-galactose pathway, through which l-galactonate was epimerized at the C5 position by the sequential activity of two dehydrogenases, resulting in d-altronate. Furthermore, this pathway completely overlapped with Route III of the non-phosphorylative l-fucose pathway. The 'substrate promiscuity' of d-altronate dehydratase protein(s) is significantly expanded to 'metabolic promiscuity' in the d-arabinose, sugar acid, l-fucose and l-galactose pathways.
  • Yasunori Watanabe, Chinatsu Iga, Yasuo Watanabe, Seiya Watanabe
    FEBS letters 593 11 1257 - 1266 2019年06月 [査読有り]
     
    In Azospirillum brasilense, a gram-negative nitrogen-fixing bacterium, l-arabinose is converted to α-ketoglutarate through a nonphosphorylative metabolic pathway. In the first step in the pathway, l-arabinose is oxidized to l-arabino-γ-lactone by NAD(P)-dependent l-arabinose 1-dehydrogenase (AraDH) belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Here, we determined the crystal structures of apo- and NADP-bound AraDH at 1.5 and 2.2 Å resolutions, respectively. A docking model of l-arabinose and NADP-bound AraDH and structure-based mutational analyses suggest that Lys91 or Asp169 serves as a catalytic base and that Glu147, His153, and Asn173 are responsible for substrate recognition. In particular, Asn173 may play a role in the discrimination between l-arabinose and d-xylose, the C4 epimer of l-arabinose.
  • Watanabe Y, Watanabe S, Itoh Y, Watanabe Y
    Biochemical and Biophysical Research Communications 511 1 135 - 140 2019年02月 [査読有り]
  • Seiya Watanabe, Fumiyasu Fukumori, Hisashi Nishiwaki, Yasuhiro Sakurai, Kunihiko Tajima, Yasuo Watanabe
    Scientific reports 9 1 155 - 155 2019年01月 [査読有り]
     研究論文(学術雑誌) 
    Pentoses, including D-xylose, L-arabinose, and D-arabinose, are generally phosphorylated to D-xylulose 5-phosphate in bacteria and fungi. However, in non-phosphorylative pathways analogous to the Entner-Dodoroff pathway in bacteria and archaea, such pentoses can be converted to pyruvate and glycolaldehyde (Route I) or α-ketoglutarate (Route II) via a 2-keto-3-deoxypentonate (KDP) intermediate. Putative gene clusters related to these metabolic pathways were identified on the genome of Herbaspirillum huttiense IAM 15032 using a bioinformatic analysis. The biochemical characterization of C785_RS13685, one of the components encoded to D-arabinonate dehydratase, differed from the known acid-sugar dehydratases. The biochemical characterization of the remaining components and a genetic expression analysis revealed that D- and L-KDP were converted not only to α-ketoglutarate, but also pyruvate and glycolate through the participation of dehydrogenase and hydrolase (Route III). Further analyses revealed that the Route II pathway of D-arabinose metabolism was not evolutionally related to the analogous pathway from archaea.
  • Yasuo Watanabe, Itsuki Kobayashi, Takanori Ohnaka, Seiya Watanabe
    Biotechnology Reports 18 e00250  2018年06月 [査読有り]
     研究論文(学術雑誌) 
    The gene encoding the Saccharomyces cerevisiae phospholipid deacylation enzyme, phospholipase B (ScPLB1), was successfully expressed in E. coli. The enzyme (Scplb1p) was engineered to have a histidine-tag at the C-terminal end and was purified by metal (Ni) affinity chromatography. Enzymatic properties, optimal pH, and substrate specificity were similar to those reported previously. For example, deacylation activity was observed in acidic pH in the absence of Ca2+ and was additive in neutral pH in the presence of Ca2+, and the enzyme had the same substrate priority as reported previously, with the exception of PE, suggesting that yeast phospholipase B could be produced in its native structure in bacterial cells. Scplb1p retained transacylation activity in aqueous medium, and esterified lysophosphatidylcholine with free fatty acid to form phosphatidylcholine in a non-aqueous, glycerin medium. We propose that phospholipase B could serve as an additional tool for in vitro enzyme-mediated phospholipid synthesis.
  • Seiya Watanabe, Daichi Morimoto, Fumiyasu Fukumori, Yasuo Watanabe
    Bioscience, biotechnology, and biochemistry 82 1 110 - 113 2018年01月 [査読有り]
     研究論文(学術雑誌) 
    The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.
  • Seiya Watanabe, Fumiyasu Fukumori, Mao Miyazaki, Shinya Tagami, Yasuo Watanabe
    Journal of bacteriology 199 16 e00255  2017年08月 [査読有り]
     研究論文(学術雑誌) 
    Hydroxyprolines, such as trans-4-hydroxy-l-proline (T4LHyp), trans-3-hydroxy-l-proline (T3LHyp), and cis-3-hydroxy-l-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (l-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like hypI gene from an l-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnXType I). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that Azospirillum brasilense NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the hypI gene (hypIS and hypIL, respectively) from A. brasilense NBRC 102289 are located separately from the l-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnXType IIa). A strain disrupted in the hypIS gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the hypI genes but also the hypK gene encoding Δ1-pyrroline-2-carboxylate reductase, which is involved in T3LHyp, d-proline, and d-lysine metabolism. On the other hand, the l-Hyp gene cluster of some other bacteria contained not only the AcnXType IIa gene but also two putative proline racemase-like genes (hypA1 and hypA2). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr).IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-l-proline (generally called l-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. l-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of trans-3-hydroxyl-proline and/or cis-3-hydroxyl-proline, a relatively rare l-hydroxyproline in nature. Several l-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by trans-4-hydroxy-l-proline, trans-3-hydroxy-l-proline, and/or cis-3-hydroxy-l-proline significantly differs between bacteria. The results of the present study show that several l-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s).
  • Seiya Watanabe, Kunihiko Tajima, Satoshi Fujii, Fumiyasu Fukumori, Ryotaro Hara, Rio Fukuda, Mao Miyazaki, Kuniki Kino, Yasuo Watanabe
    Scientific reports 6 38720 - 38720 2016年12月 [査読有り]
     研究論文(学術雑誌) 
    In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy)proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Δ1-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of α- to β-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3- and 4-hydroxyprolines.
  • Seiya Watanabe, Kunihiko Tajima, Kazuma Matsui, Yasuo Watanabe
    Bioscience, biotechnology, and biochemistry 80 12 2371 - 2375 2016年12月 [査読有り]
     研究論文(学術雑誌) 
    Flavin-containing opine dehydrogenase from Bradyrhizobium japonicum forms a heterooligomeric α4β4γ4 enzyme complex. An electron paramagnetic resonance spectroscopy analysis using wild-type and site-directed mutants revealed that [4Fe-4S] and [2Fe-2S] clusters bind to two different types of [Fe-S] binding sites in the γ- and α-subunits, respectively. The latter was found to be important for structural folding and enzyme catalysis.
  • Seiya Watanabe, Yuki Utsumi, Shigeki Sawayama, Yasuo Watanabe
    Bioscience, biotechnology, and biochemistry 80 11 2151 - 2158 2016年11月 [査読有り]
     研究論文(学術雑誌) 
    d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The kcat/Km value with d-arabinose (1.27 min-1 mM-1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.
  • Hiroaki Sakamoto, Kazuya Watanabe, Ayako Koto, Gaku Koizumi, Takenori Satomura, Seiya Watanabe, Shin-ichiro Suye
    Sensing and Bio-Sensing Research 4 37 - 39 2015年06月 研究論文(学術雑誌) 
    L-hydroxyproline (L-Hyp: trans-4-hydroxy- L-proline) is commonly found at high concentrations in connective tissue proteins such as collagen. It is a remarkably useful molecular marker because variation in the level of L-Hyp is associated with various diseases. Recently, the novel enzymes L-hydroxyproline epimerase and d-hydroxyproline dehydrogenase were isolated from bacteria. In this study, a novel electrochemical biosensor for l-Hyp was constructed using these two enzymes. L-hydroxyproline epimerase epimerized L-Hyp to D-hydroxyproline (D-Hyp: cis-4-hydroxy- D-proline), and D-Hyp was oxidized with the reaction catalyzed by d-hydroxyproline dehydrogenase and mediated by ferrocene. We found that the sensor could determine l-Hyp concentrations of 10-100. μM with high-selectivity.
  • Yasuo Watanabe, Kohki Kawata, Seiya Watanabe
    Journal of food science 80 6 H1418-24 - 24 2015年06月 [査読有り]
     研究論文(学術雑誌) 
    Gamma-aminobutyric acid (GABA) has many biological functions, including the inhibition of blood pressure increases and acceleration of growth hormone secretion. In this study, we discovered the utility of measuring the concentration of carbon dioxide (CO2 ) dissolved in the reaction solution, for development of a real-time and convenient technique to estimate GABA production. In addition to mochi barley bran, we examined the polished grains of three species: mochi barley (a variant of hulless barley), barley, and Japanese millet, all soaked in l-glutamic acid (l-Glu) solution at pH 4.5. We found a positive correlation between GABA and CO2 concentrations, and the production of CO2 was suppressed in the absence of l-Glu at pH 4.5. These results suggest that GABA content can be easily predicted by measuring the aqueous CO2 content using a CO2 sensor, during the process of GABA production in polished mochi barley grains and bran.
  • Seiya Watanabe, Yoshinobu Hiraoka, Shiori Endo, Yoshiaki Tanimoto, Yuzuru Tozawa, Yasuo Watanabe
    Journal of biotechnology 199 9 - 16 2015年04月 [査読有り]
     研究論文(学術雑誌) 
    Post-translational hydroxylation of the L-proline residue mainly occurs in collagen; therefore, the L-hydroxyprolines (L-Hyp) synthesized, including trans-4-hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp), are important markers for directly measuring the content of collagen in several biological samples. The most frequently used method to estimate the content of L-Hyp is high-performance liquid chromatography (HPLC), which is inconvenient. In the present study, we attempted to estimate the content of L-Hyp using coupling systems with metabolic enzymes of the T4LHyp (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and T3LHyp pathways (T3LHyp dehydratase (T3LHypD) and Δ(1)-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. We constructed a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells. Enzymological characterization revealed that the β-subunit acted as a catalytic subunit, and also that assembly with other subunit(s) improved the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of T4LHyp and T3LHyp were successfully estimated within the ranges of 0.004-1mM and 0.05-1mM, respectively, and were consistent with those determined by HPLC. This enzymatic method was used to measure the content of T4LHyp in the acid-hydrolysate of collagen, and blood plasma.
  • Seiya Watanabe, Yoshiaki Tanimoto, Hisashi Nishiwaki, Yasuo Watanabe
    PloS one 10 3 e0120349  2015年 [査読有り]
     研究論文(学術雑誌) 
    Proline racemase (ProR) is a member of the pyridoxal 5'-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.
  • Seiya Watanabe, Rui Sueda, Fumiyasu Fukumori, Yasuo Watanabe
    PloS one 10 9 e0138434  2015年 [査読有り]
     研究論文(学術雑誌) 
    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.
  • Hiroyuki Inoue, Seitaro Hashimoto, Akinori Matsushika, Seiya Watanabe, Shigeki Sawayama
    Journal of industrial microbiology & biotechnology 41 12 1773 - 81 2014年12月 [査読有り]
     研究論文(学術雑誌) 
    The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.
  • Yuhta Nomura, Atsushi Izumi, Yoshinori Fukunaga, Kensuke Kusumi, Koh Iba, Seiya Watanabe, Yoichi Nakahira, Andreas P M Weber, Akira Nozawa, Yuzuru Tozawa
    The Journal of biological chemistry 289 22 15631 - 41 2014年05月 [査読有り]
     研究論文(学術雑誌) 
    The guanosine 3',5'-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 μM relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 μM. The IC50 of ppGpp for GKpm was ∼10 μM. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.
  • Seiya Asada, Seiya Watanabe, Tatsuya Fujii, Hiroyuki Inoue, Kazuhiko Ishikawa, Shigeki Sawayama
    Applied biochemistry and biotechnology 172 6 3009 - 15 2014年03月 [査読有り]
     研究論文(学術雑誌) 
    A potent sugar sensor gene (g9105) was screened from the genomic data of the cellulase-producing fungus Acremonium cellulolyticus. The transcriptional level of g9105 in the SA49 transformant, which carried the knockdown RNA interference (RNAi) construct, was less than 10% compared with the parental YP-4 strain. The hairpin-type RNAi construct could be useful for this fungal gene knockdown. Changes in cellulase productivity and protein secretion between these two strains were not observed. The numbers of hyphal tips at subapical branching site were counted for the SA49 and YP-4 strains incubated with potato-dextrose medium at 30 °C for 4 days. The hyphal branching ratio of the SA49 strain was higher than that of the YP-4 strain. The present results suggest that the potent sugar sensor gene in A. cellulolyticus could be related with hyphal branch formation.
  • Watanabe S, Tanimoto Y, Yamauchi S, Tozawa Y, Sawayama S, Watanabe Y
    FEBS open bio 4 240 - 50 2014年 [査読有り]
     研究論文(学術雑誌) 
    trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.
  • Watanabe S, Tozawa Y, Watanabe Y
    FEBS open bio 4 617 - 26 2014年 [査読有り]
     研究論文(学術雑誌) 
    l-Ornithine cyclodeaminase (OCD) is involved in l-proline biosynthesis and catalyzes the unique deaminating cyclization of l-ornithine to l-proline via a Δ(1)-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD(+)-dependent l-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-l-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to l-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240-250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-l-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.
  • Seiya Watanabe, Daichi Morimoto, Fumiyasu Fukumori, Hiroto Shinomiya, Hisashi Nishiwaki, Miyuki Kawano-Kawada, Yuuki Sasai, Yuzuru Tozawa, Yasuo Watanabe
    The Journal of biological chemistry 287 39 32674 - 88 2012年09月 [査読有り]
     研究論文(学術雑誌) 
    L-hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Δ(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α(4)β(4)γ(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L- and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.
  • Sadat Mohammad Rezq Khattab, Seiya Watanabe, Masayuki Saimura, Tsutomu Kodaki
    Biochemical and biophysical research communications 404 2 634 - 7 2011年01月 [査読有り]
     研究論文(学術雑誌) 
    Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD(+) to NADP(+). In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.
  • Akinori Matsushika, Hiroyuki Inoue, Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino, Shigeki Sawayama
    Applied and environmental microbiology 75 11 3818 - 22 2009年06月 [査読有り]
     研究論文(学術雑誌) 
    The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.
  • Seiya Watanabe, Keisuke Makino
    The FEBS journal 276 6 1554 - 67 2009年03月 [査読有り]
     研究論文(学術雑誌) 
    Several bacteria, including Azotobacter vinelandii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. In a previous article, a gene cluster related to this pathway was identified, consisting of the genes encoding the four metabolic enzymes L-rhamnose-1-dehydrogenase (LRA1), L-rhamnono-gamma-lactonase (LRA2), L-rhamnonate dehydratase (LRA3) and L-2-keto-3-deoxyrhamnonate (L-KDR) aldolase (LRA4), by which L-rhamnose is converted into pyruvate and L-lactaldehyde, through analogous reaction steps to the well-known Entner-Doudoroff (ED) pathway. In this study, bioinformatic analysis revealed that Sphingomonas sp. possesses a gene cluster consisting of LRA1-3 and two genes of unknown function, LRA5 and LRA6. LRA5 catalyzed the NAD(+)-dependent dehydrogenation of several L-2-keto-3-deoxyacid-sugars, including L-KDR. Furthermore, the reaction product was converted to pyruvate and L-lactate by LRA6; this is different from the pathway of Azotobacter vinelandii. Therefore, LRA5 and LRA6 were assigned as the novel enzymes L-KDR 4-dehydrogenase and L-2,4-diketo-3-deoxyrhamnonate hydrolase, respectively. Interestingly, both enzymes were phylogenetically similar to L-rhamnose-1-dehydrogenase and D-2-keto-3-deoxyarabinonate dehydratase, respectively, and the latter was involved in the archeal nonphosphorylative d-arabinose pathway, which is partially analogous to the ED pathway. The introduction of LRA1-4 or LRA1-3, LRA5 and LAR6 compensated for the L-rhamnose-defective phenotype of an Escherichia coli mutant. Metabolic evolution and promiscuity between the alternative l-rhamnose pathway and other sugar pathways analogous to the ED pathway are discussed.
  • Akinori Matsushika, Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino, Hiroyuki Inoue, Katsuji Murakami, Osamu Takimura, Shigeki Sawayama
    Applied microbiology and biotechnology 81 2 243 - 55 2008年11月 [査読有り]
     研究論文(学術雑誌) 
    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.
  • Seiya Watanabe, Sommani Piyanart, Keisuke Makino
    The FEBS journal 275 20 5139 - 49 2008年10月 [査読有り]
     研究論文(学術雑誌) 
    Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated L-rhamnose metabolism. J Biol Chem283, 20372-20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [L-rhamnose 1-dehydrogenase (LRA1), L-rhamnono-gamma-lactonase (LRA2), L-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative L-rhamnose pathways, L-lactaldehyde is commonly produced from l-2-keto-3-deoxyrhamnonate and L-rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of L-lactaldehyde in fungi, we purified L-lactaldehyde dehydrogenase (LADH) from P. stipitis cells L-rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ionization-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1-4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, L-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase superfamily, PsLADH is poorly related to the known bacterial LADHs, including that of Escherichia coli (EcLADH). However, despite its involvement in different L-rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for L-lactaldehyde between fungal and bacterial LADHs have been acquired independently.
  • Seiya Watanabe, Masayuki Saimura, Keisuke Makino
    The Journal of biological chemistry 283 29 20372 - 82 2008年07月 [査読有り]
     研究論文(学術雑誌) 
    The Entner-Doudoroff (ED) pathway is a classic central pathway of d-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-gamma-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.
  • 渡辺隆司, 小瀧努, 牧野圭祐, 中村嘉利, 本田与一, 渡邊崇人, 渡邊誠也, 佐々木千鶴
    京都大学生存基盤科学研究ユニット研究成果報告書 2007 53 - 58 2008年06月 研究論文(その他学術会議資料等)
  • Akinori Matsushika, Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino, Shigeki Sawayama
    Journal of bioscience and bioengineering 105 3 296 - 9 2008年03月 [査読有り]
     研究論文(学術雑誌) 
    We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.
  • Yuhya Sato, Seiya Watanabe, Naoto Yamaoka, Yasuhiro Takada
    Extremophiles : life under extreme conditions 12 1 107 - 17 2008年01月 [査読有り]
     研究論文(学術雑誌) 
    The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme.
  • Seiya Watanabe, Keisuke Makino
    Seikagaku. The Journal of Japanese Biochemical Society 79 11 1059 - 64 2007年11月 [査読有り]
     研究論文(学術雑誌)
  • Seiya Watanabe, Ahmed Abu Saleh, Seung Pil Pack, Narayana Annaluru, Tsutomu Kodaki, Keisuke Makino
    Microbiology (Reading, England) 153 Pt 9 3044 - 3054 2007年09月 [査読有り]
     研究論文(学術雑誌) 
    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.
  • Seiya Watanabe, Ahmed Abu Saleh, Seung Pil Pack, Narayana Annaluru, Tsutomu Kodaki, Keisuke Makino
    Journal of biotechnology 130 3 316 - 9 2007年06月 [査読有り]
     研究論文(学術雑誌) 
    Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.
  • Kamakshaiah Charyulu Devarayapalli, Seung Pil Pack, Nagendra Kumar Kamisetty, Mitsuru Nonogawa, Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino
    Analytical and bioanalytical chemistry 388 4 919 - 28 2007年06月 [査読有り]
     研究論文(学術雑誌) 
    DNA chips prepared on a flat glass surface have unavoidable drawbacks when used for quantitative analysis. In an attempt to overcome this problem, we constructed an HPLC-type system suitable for quantitative analysis that enables base sequence- and T (m)-dependent DNA oligomer separation in a flow system. A small open tubular capillary column (300-mm x 100-microm I.D.) was used. The DNA oligomers used as probes had an amino group at the 5'-end and were immobilized on the inner silica surface of the capillary column which had been sequentially treated with 3-aminopropyltriethoxysilane, butyltrimethoxysilane, and disuccinimidylglutarate. Using the combination of probe-immobilized column placed in a column oven equipped with temperature gradient function, a nano-flow-controllable pump, a small sample-loading injector, and a capillary-fitted UV detector, we succeeded in separating complementary and non-complementary DNA oligomers in specific and quantitative modes. We also designed a temperature gradient strategy for efficient separation of target DNA oligomers in DNA mixture samples. Using a column carrying two different probes with similar T (m) values, their complementary target DNA oligomers were also separated and detected. The developed DNA open tubular capillary column system investigated in the present study could be further improved as an alternative tool to DNA chips to be used for the quantitative analysis of DNA or mRNA samples.
  • Narayana Annaluru, Seiya Watanabe, Seung Pil Pack, Ahmed Abu Saleh, Tsutomu Kodaki, Keisuke Makino
    Journal of biotechnology 129 4 717 - 22 2007年05月 [査読有り]
     研究論文(学術雑誌) 
    Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.
  • Naoko Shimada, Bunzo Mikami, Seiya Watanabe, Keisuke Makino
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 63 5 393 - 395 2007年05月 研究論文(学術雑誌) 
    1-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde. L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 angstrom resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 angstrom.
  • Seiya Watanabe, Seung Pil Pack, Ahmed Abu Saleh, Narayana Annaluru, Tsutomu Kodaki, Keisuke Makino
    Bioscience, biotechnology, and biochemistry 71 5 1365 - 9 2007年05月 [査読有り]
     研究論文(学術雑誌) 
    We focused on the effects of a mutation of xylose reductase from Pichia stipitis (PsXR) on xylose-to-ethanol fermentation using recombinant Saccharomyces cerevisiae transformed with PsXR and PsXDH (xylitol dehydrogenase from P. stipitis) genes. Based on inherent NADH-preferring XR and several site-directed mutagenetic studies using other aldo-keto reductase enzymes, we designed several single PsXR mutants. K270R showing decreased NADPH-preferring activity without a change in NADH-preferring activity was found to be a potent mutant. Strain Y-K270R transformed with K270R PsXR and wild-type PsXDH showed a 31% decrease in unfavorable xylitol excretion with 5.1% increased ethanol production as compared to the control in the fermentation of 15 g l(-1) xylose and 5 g l(-1) glucose.
  • Naoko Shimada, Bunzo Mikami, Seiya Watanabe, Keisuke Makino
    Acta crystallographica. Section F, Structural biology and crystallization communications 63 Pt 5 393 - 5 2007年05月 [査読有り]
     研究論文(学術雑誌) 
    L-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde. L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 A resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 A.
  • WATANABE Seiya, WATANABE Seiya, YAMADA Masaki, OHTSU Iwao, MAKINO Keisuke, MAKINO Keisuke
    Journal of Biological Chemistry 282 9 6685 - 95 2007年03月 [査読有り]
     研究論文(学術雑誌) 
    Azospirillum brasilense possesses an alternative pathway of L-arabinose metabolism in which α-ketoglutaric semialdehyde (αKGSA) dehydrogenase (KGSADH) is involved in the last step, the conversion of αKGSA to α-ketoglutarate. In the preceding studies, we identified a set of metabolic genes of the L-arabinose pathway including the KGSADH gene (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623; Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888; Watanabe, S., Shimada, N., Tajima, K., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 33521-33536). Here, we describe that A. brasilense possesses two different KGSADH isozymes from L-arabinose-related enzyme (KGSADH-I); that is, D-glucarate/D-galactarate-inducible KGSADH-II and hydroxy-L-proline- inducible KGSADH-III. They were purified homogeneously from A. brasilense cells grown on D-galactarate or hydroxy-L-proline, respectively. When compared with KGSADH-I, amino acid sequences of KGSADH-II and KGSADH-III were significantly similar but not totally identical. Physiological characterization using recombinant enzymes revealed that KGSADH-II and KGSADH-III showed similar high substrate specificity for αKGSA and different coenzyme specificity; that is, NAD+-dependent KGSADH-II and NADP+-dependent KGSADH-III. In the phylogenetic tree of the aldehyde dehydrogenase (ALDH) superfamily, KGSADH-II and KGSADH-III were poorly related to the known ALDH subclasses including KGSADH-I. On the other hand, ALDH-like ycbD protein involved in D-glucarate/D-galactarate operon from Bacillus subtilis is closely related to the methylmalonyl semialdehyde dehydrogenase subclass but not A. brasilense KGSADH isozymes. To estimate the correct function, the corresponding gene was expressed, purified, and characterized. Kinetic analysis revealed the physiological role as NADP+-dependent KGSADH. We conclude that three different types of KGSADH appeared in the bacterial evolutional stage convergently. Furthermore, even the same pathway such as L-arabinose and D-glucarate/D-galactarate metabolism also evolved by the independent involvement of KGSADH. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Nagendra Kumar Kamisetty, Seung Pil Pack, Mitsuru Nonogawa, Kamakshaiah Charyulu Devarayapalli, Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino
    Analytical and bioanalytical chemistry 387 6 2027 - 35 2007年03月 [査読有り]
     研究論文(学術雑誌) 
    Amine-modified oligodeoxynucleotides (AMO) are commonly used probe oligodeoxynucleotides for DNA microarray preparation. Two methods are currently used for AMO preparation--use of amine phosphoramidites protected by acid-labile monomethoxytrityl (MMT) groups or alkali-labile trifluoroacetyl (TFA) groups. Because conventional AMO preparation procedures have defects, for example stringent acidic conditions are required for deprotection of MMT and hydrophobic purification cannot be used for TFA-protected amino groups, conventional preparation of AMO is unlikely to result in the expected outcome. In this paper a method of AMO synthesis using modified H-phosphonate chemistry is suggested. An aliphatic diamine is coupled with a phosphonate group forming a phosphoramidate linkage to the last internucleotide phosphate of oligodeoxynucleotides. In this method dimethoxytrityl (DMT) purification steps are used and stringent acid deprotection is not required to obtain the AMO. Although the method could lead to formation of AMO diastereomers, melting-temperature and CD analysis showed for two AMO that DNA duplex formation was the same as when normal oligodeoxynucleotides were used. Also, when these AMO were used as probes for DNA microarrays the immobilization efficiency was similar to that for AMO probes prepared by conventional means using an amino-modifier unit. The hybridization performance of these AMO was better than for those prepared conventionally. The procedures suggested would be useful for preparation of efficient AMO for fabrication of DNA microarrays and DNA-based nanoparticle systems.
  • Protein engineering of xylose reductase from Pichia stipitis for improved NADH-specificity and the efficient ethanol production from xylose in recombinant Saccharomyces cerevisiae.
    Microbiology 153 9 3045 - 3055 2007年
  • Seiya Watanabe, Naoko Shimada, Kunihiko Tajima, Tsutomu Kodaki, Keisuke Makino
    The Journal of biological chemistry 281 44 33521 - 36 2006年11月 [査読有り]
     研究論文(学術雑誌) 
    Azospirillum brasiliense possesses an alternative pathway of L-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized L-arabinose-1-dehydrogenase and alpha-ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate (L-KDA) dehydratase, and L-arabinolactonase. N-terminal amino acid sequences of L-arabonate dehydratase and L-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the L-arabinose-1-dehydrogenase gene. Furthermore, the L-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative L-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. L-arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S](2+) cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. L-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. D-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to L-KDA aldolase involved in another alternative L-arabinose pathway, probably associating a unique evolutional event between the two alternative L-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in L-KDA dehydratase, which may be a candidate for such a natural mutation.
  • WATANABE Seiya, WATANABE Seiya, KODAKI Tsutomu, KODAKI Tsutomu, MAKINO Keisuke, MAKINO Keisuke
    Journal of Biological Chemistry 281 39 28876 - 88 2006年09月 [査読有り]
     研究論文(学術雑誌) 
    Azospirillum brasilense possesses an alternative pathway of L-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized L-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, α-ketoglutaric semialdehyde (αKGSA) dehydrogenase, catalyzing the conversion of αKGSA to α-ketoglutarate. αKGSA dehydrogenase was purified tentatively as a NAD+-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the L-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with αKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on L-arabinose. These results indicated that this ALDH gene was the only gene of the NAD+-preferring αKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, αKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative αKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for αKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino
    The Journal of biological chemistry 281 5 2612 - 23 2006年02月 [査読有り]
     研究論文(学術雑誌) 
    Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.
  • 渡辺隆司, 小瀧努, 牧野圭祐, 中村嘉利, 本田与一, 渡邊崇人, 渡邊誠也, 佐々木千鶴
    京都大学生存基盤科学研究ユニット研究成果報告書 2006 27 - 28 2006年 研究論文(その他学術会議資料等)
  • Crystallization and preliminary X-ray diffraction studies of xylitol dehydrogenase, a key enzyme for bioethanol production using yeast
    J. Matsui, S. Watanabe, T. Kodaki, B. Mikami, K. Makino
    20th IUBMB International Congress of Biochemistry and Molecular Biology,Kyoto Japan,2006.6.18-23 2006年 [査読有り]
  • Seiya Watanabe, Yoshiaki Yasutake, Isao Tanaka, Yasuhiro Takada
    Microbiology (Reading, England) 151 Pt 4 1083 - 1094 2005年04月 [査読有り]
     研究論文(学術雑誌) 
    To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from Azotobacter vinelandii (AvIDH) and Colwellia maris (CmIDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of AvIDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from Escherichia coli (EcIDH). All recombinant enzymes with a (His)(6)-tag attached to the N-terminal were overexpressed in the E. coli cells and purified by Ni(2+)-affinity chromatography. The catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) of the wild-type AvIDH and CmIDH were higher than those of EcIDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of CmIDH, and that the corresponding two regions of AvIDH are important for exhibiting higher catalytic activity and affinity for isocitrate than CmIDH. The relationships between the stability, catalytic activity and structural characteristics of AvIDH and CmIDH are discussed.
  • Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino
    The Journal of biological chemistry 280 11 10340 - 9 2005年03月 [査読有り]
     研究論文(学術雑誌) 
    Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).
  • Seiya Watanabe, Yasuhiro Takada
    Microbiology (Reading, England) 150 Pt 10 3393 - 403 2004年10月 [査読有り]
     研究論文(学術雑誌) 
    To investigate the mechanism of cold adaptation of isocitrate lyase (ICL; EC 4.1.3.1) from the psychrophilic bacterium Colwellia maris, Gln207 and Gln217 of this enzyme were substituted by His and Lys, respectively, by site-directed mutagenesis. His184 and Lys194 of ICL from Escherichia coli, corresponding to the two Gln residues of C. maris ICL, are highly conserved in the ICLs of many organisms and are known to be essential for catalytic function. The mutated ICLs (Cm-Q207H and Cm-Q217K, respectively) and wild-type enzymes of C. maris and E. coli (Cm-WT and Ec-WT) with His-tagged peptides were overexpressed in E. coli cells and purified to homogeneity. Thermolabile Cm-WT and mutated ICLs were susceptible to digestion with trypsin, while relatively thermostable Ec-WT was resistant to trypsin digestion, suggesting that the thermostability and resistance to tryptic digestion of the ICLs are related. Cm-Q207H and Cm-Q217K showed specific activities similar to Cm-WT at temperatures between 30 degrees C and 40 degrees C, but their activities between 10 degrees C and 25 degrees C were decreased, indicating that the two Gln residues of the C. maris ICL play important roles in its cold adaptation. Phylogenetic analysis of ICLs from various organisms revealed that the C. maris ICL can be categorized in a novel group, subfamily 3, together with several eubacterial ICLs.
  • Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino
    Nucleic acids symposium series (2004) 48 197 - 8 2004年 [査読有り]
     研究論文(学術雑誌) 
    For construct an efficient-ethanol fermentation system from xylose by using yeast, several protein-engineering of xylitol dehydrogenase (XDH) were carried out. At first, we created a NADP+-dependent XDH by multiple site-directed mutagenesis. Furthermore, we succeeded to improve the thermostability by introduction of the structural zinc atom into XDH which does not possess a second zinc atom natively. Finally, the introduction of a structural zinc atom into the NADP+-dependent XDH mutant increased the thermostability of this mutant and improved further the catalytic efficiency with NADP+.
  • Yoshiaki Yasutake, Seiya Watanabe, Min Yao, Yasuhiro Takada, Noriyuki Fukunaga, Isao Tanaka
    The Journal of biological chemistry 278 38 36897 - 904 2003年09月 [査読有り]
     研究論文(学術雑誌) 
    NADP+-dependent monomeric isocitrate dehydrogenase (IDH) from the nitrogen-fixing bacterium Azotobacter vinelandii (AvIDH) is one of members of the beta-decarboxylating dehydrogenase family and catalyzes the dehydration and decarboxylation of isocitrate to yield 2-oxoglutrate and CO2 in the Krebs cycle. We solved the crystal structure of the AvIDH in complex with cofactor NADP+ (AvIDH-NADP+ complex). The final refined model shows the closed form that has never been detected in any previously solved structures of beta-decarboxylating dehydrogenases. The structure also reveals all of the residues that interact with NADP+. The structure-based sequence alignment reveals that these residues were not conserved in any other dimeric NADP+-dependent IDHs. Therefore the NADP+ specificity of the monomeric and dimeric IDHs was independently acquired through the evolutional process. The AvIDH was known to show an exceptionally high turnover rate. The structure of the AvIDH-NADP+ complex indicates that one loop, which is not present in the Escherichia coli IDHs, reliably stabilizes the conformation of the nicotinamide mononucleotide of the bound NADP+ by forming a few hydrogen bonds, and such interactions are considered to be important for the monomeric enzyme to initiate the hydride transfer reaction immediately. Finally, the structure of the AvIDH is compared with that of other dimeric NADP-IDHs. Several structural features demonstrate that the monomeric IDHs are structurally more related to the eukaryotic dimeric IDHs than to the bacterial dimeric IDHs.
  • Y Yasutake, S Watanabe, M Yao, Y Takada, N Fukunaga, Tanaka, I
    STRUCTURE 10 12 1637 - 1648 2002年12月 研究論文(学術雑誌) 
    NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO2 in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 Angstrom crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.
  • Yoshiaki Yasutake, Seiya Watanabe, Min Yao, Yasuhiro Takada, Noriyuki Fukunaga, Isao Tanaka
    Structure (London, England : 1993) 10 12 1637 - 48 2002年12月 [査読有り]
     研究論文(学術雑誌) 
    NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.
  • Seiya Watanabe, Naoto Yamaoka, Noriyuki Fukunaga, Yasuhiro Takada
    Extremophiles : life under extreme conditions 6 5 397 - 405 2002年10月 [査読有り]
     研究論文(学術雑誌) 
    Isocitrate lyase (ICL) from Colwellia psychrerythraea, a psychrophilic bacterium, was purified and characterized. The subunit molecular mass was 64 kDa, which is larger than that of other bacterial ICLs. The optimal temperature for its activity was 25 degrees C, the value of K(m) for the substrate ( DL-isocitrate) was minimum at 15 degrees C, and the catalytic efficiency ( k(cat)/ K(m)) value was maximum at 20 degrees C. Furthermore, the enzyme was remarkably thermolabile and completely inactivated by incubation for 2 min at 30 degrees C. These features indicate that ICL from this bacterium is a typical cold-adapted enzyme. A partial amino acid sequence of the C. psychrerythraea ICL was very similar to that of the closely related psychrophile Colwellia maris. Expression of the gene encoding the C. psychrerythraea ICL was found to be induced by low temperatures and by acetate in the medium. The cold adaptation of the catalytic properties of ICL and the stimulated expression of its gene at low temperatures strongly suggest that this enzyme is important for the growth of this bacterium in a cold environment.
  • S Watanabe, N Yamaoka, Y Takada, N Fukunaga
    MICROBIOLOGY-SGM 148 8 2579 - 2589 2002年08月 研究論文(学術雑誌) 
    The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms. The analyses of amino acid content and primary structure of the C maris ICL suggested that it possessed many features of a cold-adapted enzyme. Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5'-untranslated regions (TS1 and TS2) were present, of which the 5' end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively. The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature. The induction of icl expression by low temperature took place in the C. maris cells grown on succinate as the carbon source but not acetate. Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract. These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C. maris is important for acetate utilization and cold adaptation.
  • Seiya Watanabe, Naoto Yamaoka, Yasuhiro Takada, Noriyuki Fukunaga
    Microbiology (Reading, England) 148 Pt 8 2579 - 2589 2002年08月 [査読有り]
     研究論文(学術雑誌) 
    The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms. The analyses of amino acid content and primary structure of the C. maris ICL suggested that it possessed many features of a cold-adapted enzyme. Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5'-untranslated regions (TS1 and TS2) were present, of which the 5' end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively. The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature. The induction of icl expression by low temperature took place in the C. maris cells grown on succinate as the carbon source but not acetate. Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract. These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C. maris is important for acetate utilization and cold adaptation.
  • Y Yasutake, S Watanabe, M Yao, N Fukunaga, Tanaka, I
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 57 11 1682 - 1685 2001年11月 研究論文(学術雑誌) 
    NADP(+) -dependent isocitrate dehydrogenase (E.C. 1.1.1.42; IDH) is an enzyme of the Krebs cycle and catalyzes the oxidative decarboxylation reaction from dl-isocitrate to alpha -ketoglutarate, with a concomitant reduction of the coenzyme NADP(+) to NADPH. Single crystals of monomeric IDH from Azotobacter vinelandii in complex with DL-isocitrate and Mn2+ were obtained by the hanging-drop vapour-diffusion method at room temperature. One crystal diffracted to a resolution of 2.9 Angstrom and was found to belong to the orthorhombic system; the space group was determined to be P2(1)2(1)2(1), with unit-cell parameters a = 108.4, b = 121.7, c = 129.7 Angstrom. The asymmetric unit contains two molecules of monomeric IDH, corresponding to a VM value of 2.66 Angstrom (3) Da(-1). The crystals were frozen in a capillary by a flash-cooling technique and MAD data were collected using Mn atoms as anomalous scatterers on beamline BL41XU at SPring-8, Japan. The positions of two Mn atoms binding to two independent IDH molecules were located from Bijvoet difference Patterson maps.
  • Y Yasutake, S Watanabe, M Yao, N Fukunaga, Tanaka, I
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 57 1682 - 1685 2001年11月 研究論文(学術雑誌) 
    NADP(+) -dependent isocitrate dehydrogenase (E.C. 1.1.1.42; IDH) is an enzyme of the Krebs cycle and catalyzes the oxidative decarboxylation reaction from dl-isocitrate to alpha -ketoglutarate, with a concomitant reduction of the coenzyme NADP(+) to NADPH. Single crystals of monomeric IDH from Azotobacter vinelandii in complex with DL-isocitrate and Mn2+ were obtained by the hanging-drop vapour-diffusion method at room temperature. One crystal diffracted to a resolution of 2.9 Angstrom and was found to belong to the orthorhombic system; the space group was determined to be P2(1)2(1)2(1), with unit-cell parameters a = 108.4, b = 121.7, c = 129.7 Angstrom. The asymmetric unit contains two molecules of monomeric IDH, corresponding to a VM value of 2.66 Angstrom (3) Da(-1). The crystals were frozen in a capillary by a flash-cooling technique and MAD data were collected using Mn atoms as anomalous scatterers on beamline BL41XU at SPring-8, Japan. The positions of two Mn atoms binding to two independent IDH molecules were located from Bijvoet difference Patterson maps.
  • S Watanabe, Y Takada, N Fukunaga
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 5 1095 - 1103 2001年05月 研究論文(学術雑誌) 
    Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20 degreesC and was rapidly inactivated at the temperatures above 30 degreesC, but the optimum temperature for the activity of MS was 45 degreesC. NaCl was found to protect ICL from heat inactivation above 30 degreesC, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The K-m for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the K-m for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.

書籍

  • Symbiosis
    渡邉 誠也 (担当:分担執筆, 範囲:Dye-linked flavin-containing dehydrogenase from bacteria related to plant)
    IntechOpen 2018年05月
  • The Handbook of Microbial Metabolism of Amino Acids
    渡邉 誠也 (担当:分担執筆, 範囲:Hydroxyproline Metabolism in Microorganisms)
    CABI (Centre for Agriculture and Biosciences International) 2017年04月
  • Protein Engineering
    渡邉 誠也 (担当:共編者(共編著者), 範囲:Generation of xylose-fermenting Saccharomyces cerevisiae by protein-engineering)
    IntechOpen 2012年02月
  • 日本醸造協会誌
    日本醸造協会 2009年
  • 化学と教育
    日本化学会 2008年
  • 生化学
    社団法人日本生化学会 2007年
  • バイオサイエンスとインダストリー
    財団法人バイオインダストリー協会 2007年
  • 新エネルギー最前線—環境調和型エネルギーシステムの構築を目指して—
    化学同人 2006年
  • 月刊エコインダストリー
    シーエムシー出版 2004年

作品等

  • 琵琶湖産水草を原料としたバイオエタノールの生産実証プロセスの 開発
    2009年 -2009年
  • ワンバッチ式バイオエタノール製造技術の研究開発
    2008年 -2008年
  • 代謝工学的手法による木質バイオマス由来五炭糖発酵酵母の育種
    2008年 -2008年
  • ワンバッチ式バイオエタノール製造技術の研究開発
    2007年 -2007年
  • ワンバッチ式バイオエタノール製造技術の研究開発
    2006年 -2006年
  • 新規糖代謝経路解明に基づくバイオマス–エタノール高効率変換 システムの開発
    2006年 -2006年
  • 五炭糖・六炭糖同時発酵酵母を用いたバイオマス–エタノール 高効率変換技術の開発
    2006年 -2006年
  • 微生物代謝系を用いたバイオマス-エタノール変換のための遺伝子 組み換え・タンパク質工学的研究
    2005年 -2005年

MISC

  • 渡辺誠也, 平岡芳信, 遠藤志織, 谷本佳彰, 渡部保夫 日本農芸化学会大会講演要旨集(Web) 2015 3C25P13 (WEB ONLY) 2015年03月
  • 渡辺誠也, 谷本佳彰, 山内清司, 戸澤譲, 渡部保夫 日本生化学会大会(Web) 87th 3P-180 (WEB ONLY) 2014年
  • 渡部保夫, 石橋智毅, 松井都, 渡辺誠也 New Food Industry 55 (3) 1 -8 2013年03月
  • 渡辺誠也, 谷本佳彰, 西脇寿, 戸澤譲, 渡部保夫 日本生化学会大会(Web) 86th 3T18P-04(1P-155) (WEB ONLY) 2013年
  • 渡辺誠也, 森本大地, 福森文康, 四宮博人, 西脇寿, 河田美幸, 笹井雄貴, 戸澤譲, 渡部保夫 日本生化学会大会(Web) 85th WEB ONLY 3T03-09 2012年
  • Yasuo Watanabe, Eriko Torii, Seiya Watanabe, Kousaku Maeda JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY-NIPPON SHOKUHIN KAGAKU KOGAKU KAISHI 59 (6) 291 -294 2012年 
    We investigated the production of gamma-aminobutyric acid (GABA) in bran obtained from the polishing of mules barley or mochi barley grains. This bran had high GABA production capability in glutamic acid solution in the absence of pyridoyxal phosphate. This might be because there is a high ratio of holo-type to apo-type glutamate decarboxylase in the bran from mules barley and mochi barley. (Received Oct. 25, 2011 : Accepted Mar. 6, 2012)
  • 渡部 保夫, 鳥居 枝里子, 渡辺 誠也, 前田 耕作 日本食品科学工学会誌 : Nippon shokuhin kagaku kogaku kaishi = Journal of the Japanese Society for Food Science and Technology 59 (6) 291 -294 2012年 
    各種麦類のヌカを用いて(PLP無添加で)GABA生産能を測定したところ,裸麦やもち麦を含む六条大麦に由来するヌカに高いGABA生産性を確認した.理由として,それらのヌカに含まれるGABA生産酵素(グルタミン酸脱炭酸酵素)のホロ酵素の割合が小麦や二条大麦に比べて高いことにあると推察された.これらはGABAの機能性を付与した食品を製造開発するために,利用可能な成果である.
  • 渡辺 誠也 バイオサイエンスとインダストリー = Bioscience & industry 69 (4) 294 -298 2011年07月
  • Sadat Mohammad Rezq Khattab, Seiya Watanabe, Masayuki Saimura, Magdi Mohamed Afifi, Abdel-Nasser Ahmad Zohri, Usama Mohamed Abdul-Raouf, Tsutomu Kodaki Green Energy and Technology 66 117 -122 2011年 
    Xylose reductase (XR) is one of the key enzymes for bio-ethanol production from lignocellulosic biomass. Intercellular redox imbalance, caused by different coenzyme specificity of XR and Xylitol dehydrogenase (XDH), has been thought to be one of the main factors of xylitol excretion. We previously succeeded by protein engineering to improve the ethanol production by reverse the XDH dependence from NAD+ to NADP+. In this study, we employed protein engineering to construct a novel strictly NADPH dependent XR from Pichia stipitis by site directed mutagenesis, in order to effective recycling of cofactor between XR and XDH, which subsequently reduce xylitol accumulation. Double mutant E223G/S271A showed strictly NADPH dependent with 90% of wild-type activity. © Springer 2011.
  • 渡辺 誠也 研究助成金受給者研究報告集 30 181 -184 2011年
  • 渡辺 誠也 日本醸造協会誌 = Journal of the Brewing Society of Japan 104 (7) 510 -515 2009年07月 
    農業残渣や間伐材等の非食用バイオマスからのエタノール生産にとって,その未利用の主要成分であるキシロースを発酵する酵母の育種は大きな課題の一つであり,近年,サッカロミセス属酵母へのキシロース発酵能の賦与が検討されてきた。ここではそのタンパク質工学的な育種戦略について,酵母によるキシロース発酵のキー酵素であるキシリトール脱水素酵素の補酵素特異性改変に関する著者らの研究を中心に解説していただいた。このような研究を通して酵母育種のタンパク質工学的アプローチが他の発酵分野にも進展することが期待される。
  • 松鹿昭則, 渡邉誠也, 小瀧努, 牧野圭祐, 井上宏之, 村上克治, 澤山茂樹 日本農芸化学会大会講演要旨集 2008 108 2008年03月
  • Seiya Watanabe, Seiya Watanabe, Seiya Watanabe, Keisuke Makino, Keisuke Makino, Keisuke Makino, Keisuke Makino Seikagaku 79 1059 -1064 2007年12月
  • 渡辺 誠也, 牧野 圭祐 バイオサイエンスとインダストリー = Bioscience & industry 65 (12) 600 -602 2007年12月
  • 渡邉誠也, 牧野圭祐 生化学 79 (11) 1059 -1064 2007年11月
  • 松鹿昭則, 渡邉誠也, 小瀧努, 牧野圭祐, 井上宏之, 村上克治, 澤山茂樹 日本生物工学会大会講演要旨集 59th 97 2007年08月
  • 新規アラビノース代謝経路に関与するL-2-ケト-3-デオキシアラボネート脱水酵素の結晶構造解析
    嶋田直子, 三上文三, 渡邉誠也, 小瀧努, 牧野圭祐 第30回日本分子生物学会大会第80回日本生化学会大会合同大会,横浜,2007.12.11-15 2007年 [査読有り]
  • 新規アラビノース代謝経路に関与する酵素L-2-keto-3-deoxyarabonate dehydrataseの結晶構造解析
    嶋田直子, 渡邉誠也, 小瀧努, 三上文三, 牧野圭祐 京都大学21世紀COEプログラム「環境調和型エネルギーの研究教育拠点形成」成果発表会,京都,2007.3.12-13 2007年 [査読有り]
  • バイオマス高効率変換酵素の立体構造解明および機能変換
    松井絢子, 依田香子, 渡邉誠也, 小瀧努, 三上文三, 牧野圭祐 京都大学21世紀COEプログラム「環境調和型エネルギーの研究教育拠点形成」成果発表会,京都,2007.3.12-13 2007年 [査読有り]
  • 小瀧努, 渡邉誠也, AHMED Abu Saleh, PACK Seung Pil, ANNALURU Narayana, 牧野圭祐 生化学 3P-1254 2007年
  • 嶋田直子, 三上文三, 渡邉誠也, 小瀧努, 牧野圭祐 日本結晶学会年会講演要旨集 2007 109 2007年
  • S Watanabe, T Kodaki, K Makino JOURNAL OF BIOLOGICAL CHEMISTRY その他 281 (15) 10652 -10652 2006年04月
  • 依田香子, 渡邉誠也, 小瀧努, 牧野圭祐 日本化学会講演予稿集 86th (2) 1428 2006年03月
  • Narayana Annaluru, Seiya Watanabe, Ahmed Abu Saleh, Tsutomu Kodaki, Keisuke Makino Nucleic acids symposium series (2004) 50 (50) 281 -2 2006年 [査読有り]
     
    Enzyme stability is one of the critical factors to construct an efficient biological conversion system. Xylitol dehydrogenase (XDH) from Pichia stipitis is one of the key enzymes for bio-ethanol fermentation system from xylose. Previously, we tried to improve thermostability of XDH by introduction of structural zinc into the enzyme and successfully obtained a mutant, named C4 mutant, with an increased unfolding temperature (J. Biol. Chem., 280:10340-10349, 2005). We focused on further improvement of the thermostability of XDH in this study and employed subsequent site directed mutagenesis in structural zinc binding region for stabilizing the structural zinc binding loop. Two variants (C4/F98R and C4/E101F) showed higher thermostability than C4 mutant judged by thermal inactivation of enzyme activity and thermal transition temperature.
  • Ahmed Abu Saleh, Seiya Watanabe, Narayana Annaluru, Tsutomu Kodaki, Keisuke Makino Nucleic acids symposium series (2004) 50 (50) 279 -80 2006年 [査読有り]
     
    We applied protein engineering to construct an efficient biomass-ethanol conversion system using Saccharomyces cerevisiae. Intercellular redox imbalance caused by the different coenzyme specificity of xylose reductase (XR) and xylitol dehydrogenase (XDH) has been thought to be one of the main factors of xylitol excretion. Introduction of NADH-dependant XR generated in this study reduced the xylitol excretion probably because of maintaining the intercellular redox balance. Ethanol fermentation was measured in batch culture under anaerobic conditions. The best strain R276H produced a maximum of 5.94 g/l ethanol with yield of 0.43 g/g from 5 g glucose/l plus 15 g xylose/l.
  • Seiya Watanabe, Tsutomu Kodaki, Keisuke Makino Nucleic acids symposium series (2004) 49 (49) 309 -10 2005年 [査読有り]
     
    Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46) catalyzing conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme involved in the first step of this L-arabinose metabolic pathway. The purified enzyme was preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had a high catalytic efficiency for both L-arabinose and D-galactose and that the L-arabinose-specific configuration at C3 and C4 is important for a preference of the substrate sugar. The N-terminal and internal amino acid sequences had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase and D-galactose 1-dehydrogenases.
  • Tsutomu Kodaki, Shinji Tsuji, Naoko Otani, Daihei Yamamoto, Kota Sreenivasa Rao, Seiya Watanabe, Masahiro Tsukatsune, Keisuke Makino Nucleic acids research. Supplement (2001) 3 (3) 303 -4 2003年 [査読有り]
     
    Expression of a number of genes encoding enzymes involved in phospholipid biosynthesis in yeast Saccharomyces cerevisiae is known to be repressed on the addition of myo-inositol and choline to the culture medium (inositol-choline regulation). All genes subject to this inositol-choline regulation have an octamer sequence 5'-CATRTGAA-3' in their upstream regions and those octamer sequences play an important role in this regulation. To confirm the role of the octamer sequence further, we studied the transcriptional regulation of two distinct S-adenosylmethionine synthetase genes (SAM1 and SAM2) of S. cerevisiae. Quantitative RT-PCR analysis showed that only the SAM2 gene was subject to the inositol-choline regulation, consistent with the fact that only the SAM2 gene has two octamer sequences in its upstream region. Furthermore, functional promoter analysis revealed that the proximal octamer sequence of the SAM2 gene has an essential role for this regulation.

受賞

  • 2018年09月 日本農芸化学会 2018年度中四国支部奨励賞
     機能未知タンパク質の新規アノテーション手法の開発と産業応用 
    受賞者: 渡邉 誠也

共同研究・競争的資金等の研究課題

  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 渡邉 誠也, 田嶋 邦彦, 福森 文康
     
    アコニターゼスーパーファミリーの7つのサブファミリーのうちアコニターゼX(AcnX)のみ機能が不明であったが、本研究者によりシス-3-ヒドロキシ-L-プロリン(C3LHyp)の脱水酵素であることが分かっていた。これをもとに本研究では、ESR測定、部位特異的変異、X線結晶構造解析によりAcnXの活性部位に存在するユニークな鉄配位様式と触媒メカニズムを明らかにした。また、四次構造と生物種によって異なる別のタイプのAcnXの機能を解析し、C3LHypを資化できる細菌において実際にC3LHyp代謝経路に含まれていることを明らかにした。
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2013年 -2015年 
    代表者 : 渡邉 誠也
     
    研究項目ごとに記載する。①D-ヒドロキシプロリン脱水素酵素(D-HypDH)の触媒メカニズムの解明:本年度の目標であった分子量の異なる3つのサブユニットを個別に大腸菌で発現させる系を構築した。また、3つを共発現させた結果D-HypDH活性をTween-20を添加した可溶性画分に検出でき、活性型組み換え酵素を得られる目途がついた。②Pyr4H2C脱アミノ化酵素の新たな触媒メカニズムの解明:本年度は、阻害剤ピルビン酸を水素化ホウ素ナトリウム(NaBH4)存在下で反応させ安定な共有結合中間体を形成させ、質量分析で推定活性残基であるリシン修飾の有無を解析することを目標とし、これに成功した。③細菌の3-ヒドロキシプロリン(T3LHyp)代謝経路の解明:当初予定になかった項目だが、①の項目を実施中にこれまで報告例のないT3LHypを資化できる細菌を発見した。T3LHypで生育させた細菌の無細胞抽出液中に2つの酵素活性、T3LHyp脱水酵素とΔ1-ピロリン-2-カルボン酸還元酵素、の誘導が確認された。バイオインフォマテックスの手法を駆使し、これら酵素をコードする遺伝子候補LhpH及びLhpIの選抜を行い、組み換え酵素の性質及び遺伝子破壊株の表現型を解析することで、代謝経路の同定に初めて成功した。④機能未知遺伝子のアノテーション:本年度着目したPA1252遺伝子が、上記Δ1-ピロリン-2-カルボン酸還元酵素を有することが分かった。しかし本遺伝子はT3LHypでは誘導されなかった。酵素学的性質を見ると、4-ヒドロキシプロリンの異性体であるシス-4-ヒドロキシ-L-プロリンの代謝に関わっている可能性が示唆された。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2009年 -2011年 
    代表者 : 渡邉 誠也
     
    木質バイオマスに含まれる五炭糖キシロースをサッカロミセス酵母に効率的に発酵させるために、細胞外の糖の取り込みとセンシング機構に注目した。ピキア酵母の推定糖輸送体遺伝子の網羅的解析から、六炭糖・五炭糖輸送能を有する遺伝子の同定に成功した。スクリーニングにより得られた有用野性株を宿主として、ピキア酵母五炭糖輸送体やサッカロミセス酵母糖センサー遺伝子の構成的な発現のキシロース発酵に与える影響を解析した。また、遺伝子組み換え酵母による木質バイオマスからのエタノール実証生産試験を行った。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2006年 -2008年 
    代表者 : 渡邉 誠也
     
    木質系バイオマスに含まれる五炭糖であるL-アラビノースのサッカロミセス酵母によるエタノール発酵を目指して、窒素固定細菌の一種から既知のものとは全く異なるL-アラビノース代謝経路の生化学的・分子生物学的・構造生物学的解析を行った。また、リグノセルロースバイオマス成分の1つであるL-ラムノースにおいても、本経路と類似の新規経路が酵母および細菌の一部に存在することを初めて発見した。

愛媛大学教員活動実績

教育活動(B)

担当授業科目(B01)

  • 2019, 前期, 学部, 生物化学I
  • 2019, 前期, 学部, 生物学実験(コンピュータ活用を含む。
  • 2019, 前期, 修士, 産業用酵素と生化学


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