277 results on '"Toshihisa Ohshima"'
Search Results
2. Stereospecificity of hydride transfer and molecular docking in FMN‐dependent NADH‐indigo reductase of Bacillus smithii
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Tomohiro Araki, and Toshihisa Ohshima
- Subjects
H‐NMR ,cofactor stereospecificity ,FMN‐dependent NADH‐indigo reductase ,molecular docking simulation ,Biology (General) ,QH301-705.5 - Abstract
In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase expressed by thermophilic Bacillus smithii. We performed 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations to reveal that the pro‐S hydrogen at the C4 position of the nicotinamide moiety in NADH was specifically transferred to the flavin‐N5 atom of FNM. Altogether, our findings may aid in the improvement of the indigo dyeing (Aizome) process. more...
- Published
- 2021
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Catalog
3. Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application
- Author
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Hironaga Akita, Junji Hayashi, Haruhiko Sakuraba, and Toshihisa Ohshima
- Subjects
D-amino acid ,thermostable D-amino acid dehydrogenase ,meso-diaminopimelate dehydrogenase ,stable isotope-labeled D-amino acid ,Ureibacillus thermosphaericus ,protein engineering ,Microbiology ,QR1-502 - Abstract
Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes. more...
- Published
- 2018
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4. A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3
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Ryushi Kawakami, Tatsuya Ohshida, Haruhiko Sakuraba, and Toshihisa Ohshima
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Ala racemase ,Ser racemase ,PLP-dependent enzyme ,hyperthermophilic archaea ,Pyrococcus horikoshii OT-3 ,D-amino acid ,Microbiology ,QR1-502 - Abstract
We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea. more...
- Published
- 2018
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5. Oral Administration of D-aspartate, but not of L-aspartate, Reduces Food Intake in Chicks
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Edi Erwan, Shozo Tomonaga, Taketo Ohmori, Yuta Mutaguchi, Toshihisa Ohshima, Mao Nagasawa, Shinobu Yasuo, Yoshinaga Tamura, and Mitsuhiro Furuse
- Subjects
brain ,d-aspartate ,food intake ,l-aspartate ,neonatal chick ,plasma ,Animal culture ,SF1-1100 - Abstract
In the present study, we determined the effects of oral administration of L- and D-aspartate (L-Asp and D-Asp) on food intake over a period of 2 h after the administration, as well as its effects on the concentration of L- and D-Asp in the brain and plasma. Chicks were orally administered different levels (0, 3.75, 7.5 and 15 mmol/kg body weight) of L-Asp (Experiment 1) and D-Asp (Experiment 2). Administration of several doses of L-Asp linearly increased the concentration of L-Asp, but not of D-Asp, in plasma. Oral L-Asp somewhat modified the levels of L- and D-Asp levels in the telencephalon, but not in the diencephalon. However, food intake was not significantly changed with doses of L-Asp. On the other hand, D-Asp strongly and dose-dependently inhibited food intake over a period of 2 h after the administration. Oral D-Asp clearly increased D-Asp levels in the plasma and diencephalon, but no significant changes in L-Asp were detected. Brain monoamine contents were only minimally influenced by L- or D-Asp administration. We conclude that D-Asp may act as an anorexigenic factor in the diencephalon. more...
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- 2013
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6. Electrochemical behavior of dye-linked L-proline dehydrogenase on glassy carbon electrodes modified by multi-walled carbon nanotubes
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Haitao Zheng, Leyi Lin, Yosuke Okezaki, Ryushi Kawakami, Haruhiko Sakuraba, Toshihisa Ohshima, Keiichi Takagi, and Shin-ichiro Suye
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dye-linked L-proline dehydrogenase ,electrocatalysis ,electron transfer ,multi-walled carbon nanotube ,Technology ,Chemical technology ,TP1-1185 ,Science ,Physics ,QC1-999 - Abstract
A glassy carbon electrode (GC) was modified by multi-walled carbon nanotubes (MWCNTs). The modified electrode showed a pair of redox peaks that resulted from the oxygen-containing functional groups on the nanotube surface. A recombinant thermostable dye-linked L-proline dehydrogenase (L-proDH) from hyperthermophilic archaeon (Thermococcus profundus) was further immobilized by physical adsorption. The modified electrode (GC/MWCNTs/L-proDH) exhibited an electrocatalytic signal for L-proline compared to bare GC, GC/L-proDH and GC/MWCNTs electrodes, which suggested that the presence of MWCNTs efficiently enhances electron transfer between the active site of enzyme and electrode surface. The immobilized L-proDH showed a typical Michaelis–Menten catalytic response with lower apparent constant. more...
- Published
- 2010
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7. Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
- Author
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Yuta Mutaguchi and Toshihisa Ohshima
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Biology (General) ,QH301-705.5 - Abstract
Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods for purification of this protein from L. otakiensis JCM 15040. Because no specific ligand that has high affinity for this enzyme has been identified, the purification was performed using ammonium sulfate fraction, four types of column chromatography and preparative Native-PAGE, not using an affinity column chromatography. We hope that the protocol will provide useful information for purification of an enzyme that cannot easily be purified using an affinity column chromatography. more...
- Published
- 2015
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8. Analysis of L- and D-Amino Acids Using UPLC
- Author
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Yuta Mutaguchi and Toshihisa Ohshima
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Biology (General) ,QH301-705.5 - Abstract
With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in crustaceans and mollusks. In this protocol, we describe a method for analysis of L- and D-amino acids using ultra-performance liquid chromatography (UPLC). To analyze D- and L-amino acids, the enantiomers are initially converted into diastereomers (diastereomers are stereoisomers that are not related as object and mirror image and are not enantiomers) using pre-column derivatization with o-phthaldialdehyde plus N-acylated cysteine (N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine). The resultant derivatives are fluorescent diastereomers. This is followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using UPLC, and the fluorescence is detected by a fluorescence detector included in UPLC system. Using this method, 16 kinds of D-amino acid can be analyzed. more...
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- 2014
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9. Two different alanine dehydrogenases from Geobacillus kaustophilus: Their biochemical characteristics and differential expression in vegetative cells and spores
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Miku Maeno, Taketo Ohmori, Daiki Nukada, Haruhiko Sakuraba, Takenori Satomura, and Toshihisa Ohshima
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Biophysics ,Molecular Biology ,Biochemistry ,Analytical Chemistry - Published
- 2023
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10. Stereospecificity of hydride transfer and molecular docking in FMN‐dependent NADH‐indigo reductase of Bacillus smithii
- Author
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Haruhiko Sakuraba, Tomohiro Araki, Kazunari Yoneda, and Toshihisa Ohshima
- Subjects
0301 basic medicine ,FMN Reductase ,Stereochemistry ,QH301-705.5 ,Flavin Mononucleotide ,Flavin mononucleotide ,Bacillus ,Reductase ,FMN‐dependent NADH‐indigo reductase ,Indigo Carmine ,Molecular Docking Simulation ,General Biochemistry, Genetics and Molecular Biology ,Indigo ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Stereospecificity ,Moiety ,Biology (General) ,Research Articles ,Nicotinamide ,Hydride ,NAD ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,cofactor stereospecificity ,H‐NMR ,Research Article - Abstract
In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase expressed by thermophilic Bacillus smithii. We performed 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations to reveal that the pro‐S hydrogen at the C4 position of the nicotinamide moiety in NADH was specifically transferred to the flavin‐N5 atom of FNM. Altogether, our findings may aid in the improvement of the indigo dyeing (Aizome) process., The stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase from the thermophilic bacterium Bacillus smithii was studied. Both analyses of 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations showed that the pro‐S hydrogen of NADH was specifically transferred to the flavin‐N5 atom of FNM. more...
- Published
- 2021
11. Activity enhancement of multicopper oxidase from a hyperthermophile via directed evolution, and its application as the element of a high performance biocathode
- Author
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Tatsuya Ohshida, Kosaku Horinaga, Hiroaki Sakamoto, Tatsunari Hirano, Eiichiro Takamura, Takenori Satomura, Shin-ichiro Suye, Kohei Inagaki, Haruhiko Sakuraba, and Toshihisa Ohshima
- Subjects
Chemistry ,Mutant ,Wild type ,Substrate (chemistry) ,Bioengineering ,General Medicine ,Directed evolution ,Multicopper oxidase ,Archaea ,Applied Microbiology and Biotechnology ,Combinatorial chemistry ,Hyperthermophile ,Kinetics ,Amino Acid Substitution ,Enzyme Stability ,Pyrobaculum ,Specific activity ,Oxidoreductases ,Biotechnology ,Thermostability - Abstract
Although multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) can be particularly useful in biotechnological applications, e.g., as a specific catalyst at the biocathode of biofuel cells (BFCs), owing to its high stability against extremely high temperatures and across a wide range of pH values, this application potential remains limited due to the enzyme's low catalytic activity. A directed evolution strategy was conducted to improve McoP catalytic activity, and the No. 571 mutant containing four amino acid substitutions was identified, with specific activity approximately 9-fold higher than that of the wild type enzyme. Among the substitutions, the single amino acid mutant F290I was essential in enhancing catalytic activity, with a specific activity approximately 12-fold higher than that of the wild type enzyme. F290I thermostability and pH stability were notably comparable with values obtained for the wild type. Crystal structure analysis suggested that the F290I mutant increased loop flexibility near the T1 Cu center, and affected electron transfer between the enzyme and substrate. Additionally, electric current density of the F290I mutant-immobilized electrode was 7-fold higher than that of the wild type-immobilized one. These results indicated that F290I mutant was a superior catalyst with potential in practical biotechnological applications. more...
- Published
- 2021
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12. Self-assembly of Aeropyrum pernix bacilliform virus 1 (APBV1) major capsid protein and its application as building blocks for nanomaterials
- Author
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Yuka Sumikama, Atsushi Takashima, Tomohiro Mochizuki, Haruhiko Sakuraba, Toshihisa Ohshima, Shinji Sugihara, Shin-ichiro Suye, and Takenori Satomura
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Escherichia coli ,Molecular Medicine ,Capsid Proteins ,General Medicine ,Aeropyrum ,Microbiology ,Archaea ,Recombinant Proteins ,Nanostructures - Abstract
Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications. more...
- Published
- 2022
13. Structural and biochemical characterization of an extremely thermostable FMN-dependent NADH-indigo reductase from Bacillus smithii
- Author
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Misa Yoshioka, Tomohiro Araki, Toshihisa Ohshima, Kazunari Yoneda, and Haruhiko Sakuraba
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FMN Reductase ,Flavin Mononucleotide ,Bacillus ,02 engineering and technology ,Reductase ,Indigo Carmine ,medicine.disease_cause ,Biochemistry ,Catalysis ,Indigo ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,Escherichia coli ,medicine ,NADH, NADPH Oxidoreductases ,Cloning, Molecular ,Molecular Biology ,030304 developmental biology ,Thermostability ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Thermophile ,Active site ,General Medicine ,NAD ,021001 nanoscience & nanotechnology ,Molecular Docking Simulation ,Kinetics ,Enzyme ,Indigo carmine ,Mutagenesis, Site-Directed ,biology.protein ,Oxidoreductases ,0210 nano-technology - Abstract
The FMN-dependent NADH-indigo reductase gene from the thermophilic bacterium Bacillus smithii was overexpressed in Escherichia coli. The expressed enzyme functioned as a highly thermostable indigo reductase that retained complete activity even after incubation at 100 °C for 10 min. Furthermore, B. smithii indigo reductase exhibited high stability over a wider pH range and longer storage periods compared with indigo reductases previously identified from other sources. The enzyme catalyzed the reduction of various azo compounds and indigo carmine. The crystal structures of the wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and the Y151F mutant enzyme in complex with FMN were determined by the molecular replacement method and refined at resolutions of 1.97 and 1.95 A, respectively. Then, indigo carmine molecule was modeled into the active site using the molecular docking simulation and the binding mode of indigo carmine was elucidated. In addition, the structure of B. cohnii indigo reductase, which is relatively less stable than B. smithii indigo reductase, was constructed by homology modeling. The factor contributing to the considerably higher thermostability of B. smithii indigo reductase was analyzed by comparing its structure with that of B. cohnii indigo reductase, which revealed that intersubunit aromatic interactions (F105-F172′ and F172-F105′) may be responsible for the high thermostability of B. smithii indigo reductase. Notably, site-directed mutagenesis results showed that F105 plays a major role in the intersubunit aromatic interaction. more...
- Published
- 2020
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14. NADP
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Toshihisa, Ohshima, Masaki, Tanaka, and Taketo, Ohmori
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Kinetics ,Pseudomonas ,Escherichia coli ,NADPH Dehydrogenase ,Amino Acid Oxidoreductases ,Arginine ,NADP ,Substrate Specificity - Abstract
l-Arginine dehydrogenase (L-ArgDH) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of l-arginine to the oxo analog in the presence of NAD(P)
- Published
- 2022
15. Complete Genome Sequence of Bacillus cereus Strain HT18, Isolated from Forest Soil
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Minami Matsunaka, Nguyen Cong Thanh, Tatsuya Uedoi, Takashi Iida, Yasuhiro Fujino, Taketo Ohmori, Yasuaki Hiromasa, Toshihisa Ohshima, and Katsumi Doi
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Immunology and Microbiology (miscellaneous) ,Genetics ,Molecular Biology - Abstract
The genome sequence of Bacillus cereus strain HT18, isolated from forest soil, was 5,333,415 bp long. The genome included 5,825 putative coding sequences and 35.2% GC content; the strain had 5 plasmids. Average nucleotide identity based on BLAST+ (ANIb) and digital DNA-DNA hybridization (dDDH) results showed that HT18 was 98.78% and 90.70% homologous, respectively, to B. cereus ATCC 14579 T . more...
- Published
- 2022
16. Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection
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Takenori Satomura, Kohei Uno, Norio Kurosawa, Haruhiko Sakuraba, Toshihisa Ohshima, and Shin-ichiro Suye
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QH301-705.5 ,Archaeal Proteins ,l<%2Fspan>-lactate+dehydrogenase%22">dye-linked l-lactate dehydrogenase ,FMN ,hyperthermophilic archaeon ,thermostable enzyme ,heterogeneous expression ,Gene Expression ,Biosensing Techniques ,Catalysis ,Article ,Inorganic Chemistry ,Electron Transport ,dye-linked l-lactate dehydrogenase ,Enzyme Stability ,Biology (General) ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,Spectroscopy ,L-Lactate Dehydrogenase ,Organic Chemistry ,Temperature ,General Medicine ,Recombinant Proteins ,Computer Science Applications ,Chemistry ,Protein Subunits ,Multigene Family ,Sulfolobaceae ,Protein Multimerization ,Oxidation-Reduction - Abstract
Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH. more...
- Published
- 2021
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17. Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii
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Ryushi Kawakami, Tatsuya Ohshida, Junji Hayashi, Kazunari Yoneda, Toshio Furumoto, Toshihisa Ohshima, and Haruhiko Sakuraba
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Models, Molecular ,Ornithine ,Structural Biology ,Pyridoxal Phosphate ,Humans ,General Medicine ,Pyrococcus horikoshii ,Crystallography, X-Ray ,Molecular Biology ,Biochemistry ,Archaea ,Transaminases ,Substrate Specificity - Abstract
Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT. more...
- Published
- 2021
18. Separator-less Amino Acid-powered Biofuel Cell Through an Artificial Multi-enzyme Cascade Reaction
- Author
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T Satomura, Kousaku Horinaga, Eiichiro Takamura, Hiroaki Sakamoto, Haruhiko Sakuraba, Toshihisa Ohshima, and Shin-ichiro Suye
- Abstract
Objective This study was aimed at constructing a highly stable one-compartment enzymatic biofuel cell (EFC) without a separator through a multi-enzyme cascade reaction pathway. Results A separator-less EFC composed of a multi-enzyme cascade anode containing four dehydrogenases from a thermophile and a cathode devised using a multi-copper oxidase mutant with enhanced enzyme activity from a hyperthermophile was developed. To fabricate an EFC without a separator, redox mediators utilized in the enzymatic cascade reaction were also immobilized on the anode. In the presence of the fuel 100 mM L -proline, the separator-less EFC with four thermophilic dehydrogenase-modified anode achieved a maximum power density of 11.3 υW/cm 2 at 37°C, which was 1.6-fold higher than that of a similar EFC fabricated with a one enzyme-modified anode. The separator-less EFC composed of a multi-enzyme modified anode maintained approximately 57% of the load current at 0.3 V measured on the first day of EFC fabrication, even after 4 days.Conclusion Efficient L-proline electric generation utilizing a separator-less EFC composed of a multi-enzyme modified anode through a multi-step fuel oxidation reaction and a highly stable multi-copper oxidase mutant-modified cathode was successfully achieved over a long period. The long-term stability of the separator-less EFC can facilitate its application as an efficient power source for implantable medical devices requiring continuous operation. more...
- Published
- 2021
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19. NADP+-dependent l-arginine dehydrogenase from Pseudomonas velonii: Purification, characterization and application to an l-arginine assay
- Author
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Toshihisa Ohshima, Masaki Tanaka, and Taketo Ohmori
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Biotechnology - Published
- 2022
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20. Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay
- Author
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Toshihisa Ohshima, Taketo Ohmori, and Masaki Tanaka
- Subjects
chemistry.chemical_classification ,Biochemistry ,chemistry ,Arginine ,Dehydrogenase ,Arginine dehydrogenase ,Amino acid - Abstract
Purpose: The primary aim of this study was the purification and characterization of an NADP-dependent L arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) as a novel amino acid dehydrogenase from Pseudomonas veronii. We then applied the enzyme to an L-arginine assay. Methods: An L-ArgDH gene from P. veronii JCM11942 was amplified by PCR using primers based on the N and C-terminal sequences inferred from a putative L-ArgDH gene (PverR02_12350) found in the P. veronii genome. The L-ArgDH activity of the product expressed in Escherichia coli was confirmed, after which the enzyme was purified, characterized, and applied to an L-Arg microassay. Results: The P. veroniiJCM11942 gene was expressed in E. coli, and the gene product exhibited strong NADP dependent L-ArgDH activity. The crude enzyme was unstable but was stabilized by the presence of 10% glycerol under neutral pH conditions. The enzyme was purified to homogeneity through a single Ni-chelate affinity chromatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed L-arginine oxidation in the presence of NADP, with maximal activity at pH 9.5. The apparent Km values for L-arginine and NADP were 2.5 and 0.21 mM, respectively. A simple colorimetric microassay for L-arginine was achieved using the enzyme. Conclusions: The L-ArgDH gene from P. veronii JCM 11942 was successively expressed in E. coli. The product exhibited NADP-dependent L-ArgDH dehydrogenase activity, and the enzyme was purified and characterized as a novel amino acid dehydrogenase. Furthermore, a simple colorimetric assay for L-arginine using L-ArgDH was achieved. Conflict of interest: The authors declare that they have no competing interests. more...
- Published
- 2021
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21. Unique active site formation in a novel galactose 1‐phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Junji Hayashi, and Tatsuya Ohshida
- Subjects
Models, Molecular ,Protein Conformation, alpha-Helical ,Hot Temperature ,Gene Expression ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Structural Biology ,Enzyme Stability ,Moiety ,Cloning, Molecular ,chemistry.chemical_classification ,0303 health sciences ,biology ,Galactosephosphates ,030302 biochemistry & molecular biology ,Recombinant Proteins ,Protein Binding ,Archaeal Proteins ,Protein subunit ,Genetic Vectors ,03 medical and health sciences ,Escherichia coli ,medicine ,Humans ,UTP-Hexose-1-Phosphate Uridylyltransferase ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Molecular Biology ,Gene ,030304 developmental biology ,Binding Sites ,Sequence Homology, Amino Acid ,Active site ,Substrate (chemistry) ,Kinetics ,Protein Subunits ,Enzyme ,chemistry ,Galactose ,Mutation ,Pyrobaculum ,biology.protein ,Protein Conformation, beta-Strand ,Protein Multimerization ,Sequence Alignment - Abstract
A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction. more...
- Published
- 2019
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22. Improvement in Electron Transfer Efficiency Between Multicopper Oxidase and Electrode by Immobilization of Directly Oriented Enzyme Molecules
- Author
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Hiroaki Sakamoto, Takuto Nakamura, Takenori Satomura, Eiichiro Takamura, Shin-ichiro Suye, Haruhiko Sakuraba, Toshihisa Ohshima, and Haruto Suzuki
- Subjects
chemistry.chemical_classification ,Electron transfer ,Enzyme ,Polymers and Plastics ,Chemistry ,Materials Science (miscellaneous) ,Electrode ,Chemical Engineering (miscellaneous) ,Molecule ,Multicopper oxidase ,Photochemistry ,Industrial and Manufacturing Engineering - Published
- 2019
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23. Construction of a novel bioanode for amino acid powered fuel cells through an artificial enzyme cascade pathway
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Shin-ichiro Suye, Haruhiko Sakuraba, Hiroaki Sakamoto, Takenori Satomura, Toshihisa Ohshima, Shino Tanaka, Kousaku Horinaga, and Eiichiro Takamura
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0106 biological sciences ,0301 basic medicine ,Proline ,Bioelectric Energy Sources ,Bioengineering ,Dehydrogenase ,Nicotinamide adenine dinucleotide ,01 natural sciences ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,chemistry.chemical_compound ,Electricity ,Cascade reaction ,Multienzyme Complexes ,010608 biotechnology ,Electrodes ,biology ,Artificial enzyme ,Thermophile ,NADH dehydrogenase ,General Medicine ,Combinatorial chemistry ,030104 developmental biology ,chemistry ,Ferrocene ,biology.protein ,NAD+ kinase ,Oxidoreductases ,Oxidation-Reduction ,Biotechnology - Abstract
The construction of a novel bioanode based on l-proline oxidation using a cascade reaction pathway comprised of thermostable dehydrogenases. A novel multi-enzymatic cascade pathway, containing four kinds of dehydrogenases from thermophiles (dye-linked l-proline dehydrogenase, nicotinamide adenine dinucleotide (NAD)-dependent Δ1-pyrroline-5-carboxylate dehydrogenase, NAD-dependent l-glutamate dehydrogenase and dye-linked NADH dehydrogenase), was designed for the generation of six-electrons from one molecule of l-proline. The current density of the four-dehydrogenase-immobilized electrode, with a voltage of + 450 mV (relative to that of Ag/AgCl), was 226.8 μA/cm2 in the presence of 10 mM l-proline and 0.5 mM ferrocene carboxylate at 50 °C. This value was 4.2-fold higher than that of a similar electrode containing a single dehydrogenase. In addition, about 54% of the initial current in the multi-enzyme cascade bioanode was maintained even after 15 days. Efficient deep oxidation of l-proline by multiple-enzyme cascade reactions was achieved in our designed electrode. The multi-enzyme cascade bioanode, which was built using thermophilic dehydrogenases, showed high durability at room temperature. The long-term stability of the bioanode indicates that it shows great potential for applications as a long-lived enzymatic fuel cell. more...
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- 2019
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24. Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis
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Junji Hayashi, Chinatsu Kinoshita, Haruhiko Sakuraba, Mikio Sato, Toshihisa Ohshima, Ryushi Kawakami, and Tomoki Kawase
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0301 basic medicine ,Archaeal Proteins ,030106 microbiology ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,Genes, Archaeal ,Substrate Specificity ,03 medical and health sciences ,Pyrococcus horikoshii ,Amino Acid Sequence ,Amino-acid racemase ,Thermococcus litoralis ,Molecular Biology ,Peptide sequence ,Phylogeny ,Amino Acid Isomerases ,chemistry.chemical_classification ,biology ,Organic Chemistry ,pyridoxal 5ʹ-phosphate ,Amino-acid racemase activity ,General Medicine ,biology.organism_classification ,Enzyme assay ,Amino acid ,Thermococcus ,030104 developmental biology ,Enzyme ,chemistry ,amino acid racemase ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Biotechnology ,Molecular Chaperones - Abstract
The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea. more...
- Published
- 2021
25. Site-Directed Mutagenesis of Multicopper Oxidase from Hyperthermophilic Archaea for High-Voltage Biofuel Cells
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Shin-ichiro Suye, Shunsuke Taki, Eiichiro Takamura, Haruhiko Sakuraba, Takenori Satomura, Toshihisa Ohshima, and Hiroaki Sakamoto
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0106 biological sciences ,Bioelectric Energy Sources ,Archaeal Proteins ,Mutation, Missense ,Bioengineering ,Multicopper oxidase ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,Redox ,Catalysis ,chemistry.chemical_compound ,010608 biotechnology ,Site-directed mutagenesis ,Molecular Biology ,chemistry.chemical_classification ,ABTS ,biology ,010405 organic chemistry ,Ligand ,General Medicine ,biology.organism_classification ,Combinatorial chemistry ,0104 chemical sciences ,Enzyme ,chemistry ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,Pyrobaculum ,Oxidoreductases ,Biotechnology ,Archaea - Abstract
Enzymes from hyperthermophilic archaea are potential candidates for industrial use because of their superior pH, thermal, and long-term stability, and are expected to improve the long-term stability of biofuel cells (BFCs). However, the reported multicopper oxidase (MCO) from hyperthermophilic archaea has lower redox potential than MCOs from other organisms, which leads to a decrease in the cell voltage of BFCs. In this study, we attempted to positively shift the redox potential of the MCO from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP). Mutations (M470L and M470F) were introduced into the axial ligand of the T1 copper atom of McoP, and the enzymatic chemistry and redox potentials were compared with that of the parent (M470). The redox potentials of M470L and M470F shifted positively by about 0.07 V compared with that of M470. In addition, the catalytic activity of the mutants towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) increased 1.2–1.3-fold. The thermal stability of the mutants and the electrocatalytic performance for O2 reduction of M470F was slightly reduced compared with that of M470. This research provides useful enzymes for application as biocathode catalysts for high-voltage BFCs. more...
- Published
- 2020
26. Characterization of dye-linked d-amino acid dehydrogenase from Sulfurisphaera tokodaii expressed using an archaeal recombinant protein expression system
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Toshihisa Ohshima, Shin-ichiro Suye, Norio Kurosawa, Takenori Satomura, Haruhiko Sakuraba, and Shin Emoto
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0106 biological sciences ,0301 basic medicine ,Sulfolobus acidocaldarius ,D-Amino-Acid Oxidase ,Gene Expression ,Bioengineering ,Dehydrogenase ,D-amino acid dehydrogenase ,medicine.disease_cause ,01 natural sciences ,Applied Microbiology and Biotechnology ,Sulfolobus ,Gene product ,03 medical and health sciences ,010608 biotechnology ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,Escherichia coli ,Peptide sequence ,chemistry.chemical_classification ,Archaea ,Recombinant Proteins ,Amino acid ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Biotechnology - Abstract
A gene encoding a dye-linked d -amino acid dehydrogenase (Dye-DADH) homologue was found in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. The predicted amino acid sequence suggested that the gene product is a membrane-bound type enzyme. The gene was overexpressed in Escherichia coli, but the recombinant protein was exclusively produced as an inclusion body. In order to avoid production of the inclusion body, an expression system using the thermoacidophilic archaeon Sulfolobus acidocaldarius instead of E. coli as the host cell was constructed. The gene was successfully expressed in Sulfolobus acidocaldarius, and its product was purified to homogeneity and characterized. The purified enzyme catalyzed the dehydrogenation of various d -amino acids, with d -phenylalanine being the most preferred substrate. The enzyme retained its full activity after incubation at 90 °C for 30 min and after incubation at pH 4.0–11.0 for 30 min at 50 °C. This is the first report on membrane-bound Dye-DADH from thermophilic archaea that was successfully expressed in an archaeal host. more...
- Published
- 2020
27. A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima
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Haruhiko Sakuraba, Toshihisa Ohshima, Kazunari Yoneda, Kohei Koba, Tatsuya Ohshida, Taketo Ohmori, and Junji Hayashi
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Threonine ,0301 basic medicine ,Hot Temperature ,Protein Conformation ,Dehydrogenase ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme Stability ,Escherichia coli ,Thermotoga maritima ,Aspartate kinase ,Phosphofructokinase 2 ,Amino Acid Sequence ,Bifunctional ,Molecular Biology ,Homoserine dehydrogenase ,Aspartic Acid ,Sequence Homology, Amino Acid ,030102 biochemistry & molecular biology ,biology ,Organic Chemistry ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Hyperthermophile ,Kinetics ,030104 developmental biology ,chemistry ,Aspartokinase Homoserine Dehydrogenase ,Biocatalysis ,Electrophoresis, Polyacrylamide Gel ,Bacteria ,Biotechnology - Abstract
The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. l-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by l-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to l-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T). more...
- Published
- 2018
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28. Mechanism of gamma-aminobutyric acid (GABA) production by a lactic acid bacterium in yogurt-sake
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Taketo Ohmori, Toshihisa Ohshima, and Manaka Tahara
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0106 biological sciences ,0301 basic medicine ,Streptococcus thermophilus ,Glutamate decarboxylase ,Bioengineering ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,gamma-Aminobutyric acid ,03 medical and health sciences ,chemistry.chemical_compound ,010608 biotechnology ,medicine ,Food science ,biology ,Glutamate decarboxylase activity ,food and beverages ,biology.organism_classification ,Lactic acid ,030104 developmental biology ,Lactic acid bacterium ,nervous system ,chemistry ,Fermentation ,Bacteria ,medicine.drug - Abstract
We examined the gamma-aminobutyric acid (GABA) content of various kinds of fermented alcoholic beverages, including sake, shochu and yogurt-sake, and found that yogurt-sake contains a high concentration of GABA, higher than the sum of the concentrations in the sake and yogurt starting materials. To investigate the GABA production mechanism, we isolated the lactic acid bacteria (LAB) probably responsible for GABA production in the liquor and identified Streptococcus thermophilus Hp as a high GABA-producer and S. thermophilus Lp as a low GABA-producer. Cell extract from S. thermophilus Hp showed much higher glutamate decarboxylase activity than that from S. thermophilus Lp. During production of yogurt-sake using commercially available sake and yogurt produced with S. thermophilus Hp, the GABA increased over time, while the l -Glu was almost completely consumed. Moreover, by using sake supplemented with l -Glu, we successfully elevated the GABA concentration in yogurt-sake. By contrast, no GABA production was detected in yogurt-sake prepared using S. thermophilus Lp. These results indicate that LAB with highly active glutamate decarboxylase is responsible for GABA production in yogurt-sake. Our findings suggest GABA-enriched drinks are easily produced using yogurt and provide potentially useful information for production of other GABA-enriched foods. more...
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- 2018
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29. Enzymological characteristics of a novel archaeal dye-linked d-lactate dehydrogenase showing loose binding of FAD
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Toshihisa Ohshima, Takenori Satomura, Junji Hayashi, Shin-ichiro Suye, Haruhiko Sakuraba, and Tatsuya Ohshida
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0301 basic medicine ,Archaeal Proteins ,Tenax ,Sulfolobus tokodaii ,Dehydrogenase ,Biosensing Techniques ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,medicine ,Aeropyrum pernix ,Lactate Dehydrogenases ,Escherichia coli ,Peptide sequence ,Thermoproteus ,Sequence Homology, Amino Acid ,biology ,Chemistry ,Molecular Mimicry ,Archaeoglobus fulgidus ,Electrochemical Techniques ,General Medicine ,biology.organism_classification ,030104 developmental biology ,Biochemistry ,Structural Homology, Protein ,Flavin-Adenine Dinucleotide ,D-lactate dehydrogenase ,bacteria ,Molecular Medicine - Abstract
A gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD. more...
- Published
- 2018
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30. Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction
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Toshihisa Ohshima, Hirokazu Suzuki, Tomoaki Abe, and Katsumi Doi
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DNA, Bacterial ,0301 basic medicine ,Dinitrocresols ,030106 microbiology ,Bacillus ,Flavoprotein ,Reductase ,Indigo Carmine ,Applied Microbiology and Biotechnology ,Catalysis ,Indigo ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Alkaliphile ,NADH, NADPH Oxidoreductases ,Amino Acid Sequence ,Coloring Agents ,Phylogeny ,chemistry.chemical_classification ,Base Sequence ,Flavoproteins ,biology ,Sequence Analysis, DNA ,General Medicine ,Nitroreductases ,NAD ,biology.organism_classification ,Bacterial Typing Techniques ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Indigo carmine ,biology.protein ,ATP-Binding Cassette Transporters ,Polygonum ,Dyeing ,Biotechnology - Abstract
Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were more...
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- 2018
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31. Crystal structure of the NADP+ and tartrate-bound complex of l-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis
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Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, and Tomohiro Araki
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0301 basic medicine ,chemistry.chemical_classification ,biology ,Stereochemistry ,Chemistry ,Substrate (chemistry) ,Active site ,Dehydrogenase ,General Medicine ,Protein engineering ,Substrate analog ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,biology.protein ,Molecular Medicine ,Binding site ,Peptide sequence - Abstract
A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP+/sulfate ion at 1.18 A and the structure in complex with NADP+/L-tartrate (substrate analog) at 1.57 A. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP+ and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering. more...
- Published
- 2018
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32. First characterization of archaeal amino acid racemase
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Haruhiko Sakuraba, Toshihisa Ohshima, Taketo Ohmori, and Ryushi Kawakami
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0301 basic medicine ,030106 microbiology ,Bioengineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Substrate Specificity ,broad substrate specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Pyrococcus horikoshii ,Aromatic amino acids ,medicine ,Amino Acid Sequence ,Amino-acid racemase ,Escherichia coli ,Amino Acid Isomerases ,Thermostability ,chemistry.chemical_classification ,biology ,Temperature ,hyperthermophilic archaeon ,biology.organism_classification ,Hyperthermophile ,Amino acid ,Pyrococcus horikoshii OT-3 ,Enzyme ,chemistry ,Biochemistry ,kinetics ,amino acid racemase ,Hydrophobic and Hydrophilic Interactions ,Biotechnology - Abstract
A novel amino acid racemase with broad substrate specificity (BAR) was recently isolated from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Characterization of this enzyme has been difficult, however, because the recombinant enzyme is produced mainly as an inclusion body in Escherichia coli. In this study, expression of the recombinant protein into the soluble fraction was markedly improved by co-expression with chaperone molecules. The purified enzyme retained its full activity after incubation at 80°C for at least 2 h in buffer (pH 7-10), making this enzyme the most thermostable amino acid racemase so far known. Besides the nine amino acids containing hydrophobic and aromatic amino acids previously reported (Kawakami et al., Amino Acids, 47, 1579-1587, 2015), the enzyme exhibited substantial activity toward Thr (about 42% of relative activity toward Phe) and showed no activity toward Arg, His, Gln, and Asn. The substrate specificity of this enzyme thus differs markedly from those of other known amino acid racemases. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. The high reactivity toward Trp and Tyr, as well as extremely high thermostability, is likely a major advantage of using BAR for biochemical conversion of these aromatic amino acids. more...
- Published
- 2017
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33. Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase fromLactobacillus buchneri
- Author
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Yuta Mutaguchi, Toshihisa Ohshima, Junji Hayashi, Taketo Ohmori, Yume Minemura, Haruhiko Sakuraba, Kazunari Yoneda, and Noriko Nakagawa
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Isomerase ,Crystallography, X-Ray ,03 medical and health sciences ,Tetramer ,Structural Biology ,Amino Acid Sequence ,Isoleucine ,Amino-acid racemase ,Amino Acid Isomerases ,Lactobacillus buchneri ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Substrate (chemistry) ,Active site ,biology.organism_classification ,Lactobacillus ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Pyridoxal Phosphate ,biology.protein ,Sequence Alignment - Abstract
Crystal structures ofLactobacillus buchneriisoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5′-phosphate (PLP), in complex withN-(5′-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex withN-(5′-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of theL. buchneriisoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-∊-caprolactam racemase fromAchromobactor obaeand γ-aminobutyrate aminotransferase fromEscherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved inL. buchneriisoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine. more...
- Published
- 2017
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34. First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase
- Author
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Haruhiko Sakuraba, Tatsuya Ohshida, Takenori Satomura, Ryushi Kawakami, Toshihisa Ohshima, and Junji Hayashi
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0301 basic medicine ,archaea ,Archaeal Proteins ,030106 microbiology ,Sodium Chloride ,medicine.disease_cause ,aldehyde ,Haloarcula japonica ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme Stability ,Escherichia coli ,medicine ,Aldehyde-Lyases ,Thermostability ,chemistry.chemical_classification ,Haloarcula ,2-deoxy-D-ribose-5-phosphate aldolase ,biology ,Aldolase A ,Acetaldehyde ,biology.organism_classification ,Recombinant Proteins ,Halophile ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,Biocatalysis ,biology.protein ,organic solvent ,halophile ,Biotechnology - Abstract
2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA. more...
- Published
- 2016
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35. Unique coenzyme binding mode of hyperthermophilic archaealsn-glycerol-1-phosphate dehydrogenase fromPyrobaculum calidifontis
- Author
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Kazunari Yoneda, Haruhiko Sakuraba, Toshihisa Ohshima, Kaori Yamamoto, and Junji Hayashi
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0301 basic medicine ,chemistry.chemical_classification ,biology ,030106 microbiology ,Dehydrogenase ,Biochemistry ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,chemistry ,Structural Biology ,Oxidoreductase ,Ribose ,biology.protein ,Coenzyme binding ,NADPH binding ,NAD+ kinase ,Molecular Biology - Abstract
A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (Vmax ) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 A. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc. more...
- Published
- 2016
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36. Catalytic properties and crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from the hyperthermophilic archaeon Aeropyrum pernix K1
- Author
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Tomohiro Araki, Kazunari Yoneda, Yudai Fukuda, Toshihisa Ohshima, and Haruhiko Sakuraba
- Subjects
Models, Molecular ,0301 basic medicine ,Carbonyl Reductase ,Protein Conformation ,Archaeal Proteins ,Aeropyrum ,Bioengineering ,Crystallography, X-Ray ,Applied Microbiology and Biotechnology ,Biochemistry ,Genes, Archaeal ,Avian Proteins ,03 medical and health sciences ,Species Specificity ,Oxidoreductase ,Catalytic Domain ,Enzyme Stability ,Animals ,Aeropyrum pernix ,Amino Acid Sequence ,Cloning, Molecular ,Thermostability ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,030102 biochemistry & molecular biology ,biology ,biology.organism_classification ,Recombinant Proteins ,Hyperthermophile ,Alcohol Oxidoreductases ,Kinetics ,030104 developmental biology ,Enzyme ,chemistry ,Mutagenesis, Site-Directed ,NAD+ kinase ,Chickens ,Biotechnology - Abstract
A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli. Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10min up to 90°C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher Km values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme's lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR. more...
- Published
- 2016
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37. Moss Chloroplasts Are Surrounded by a Peptidoglycan Wall Containing D-Amino Acids
- Author
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Yasuhiro Shimizu, Koji Tanidokoro, Takayuki Hirano, Susumu Takio, Hayato Ishikawa, Toshihisa Ohshima, Hiroyoshi Takano, Momo Sato, Yutaka Kawarabayasi, Katsuaki Takechi, and Shinji Tadano
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0106 biological sciences ,0301 basic medicine ,Chloroplasts ,Mutant ,Bryophyta ,Peptidoglycan ,Plant Science ,Breakthrough Report ,Biology ,Physcomitrella patens ,01 natural sciences ,Bacterial cell structure ,03 medical and health sciences ,chemistry.chemical_compound ,otorhinolaryngologic diseases ,Arabidopsis thaliana ,Plastid ,chemistry.chemical_classification ,food and beverages ,Cell Biology ,biology.organism_classification ,Bryopsida ,Amino acid ,Chloroplast ,030104 developmental biology ,Biochemistry ,chemistry ,010606 plant biology & botany - Abstract
It is believed that the plastids in green plants lost peptidoglycan (i.e., a bacterial cell wall-containing d-amino acids) during their evolution from an endosymbiotic cyanobacterium. Although wall-like structures could not be detected in the plastids of green plants, the moss Physcomitrella patens has the genes required to generate peptidoglycan (Mur genes), and knocking out these genes causes defects in chloroplast division. Here, we generated P. patens knockout lines (∆Pp-ddl) for a homolog of the bacterial peptidoglycan-synthetic gene encoding d-Ala:d-Ala ligase. ∆Pp-ddl had a macrochloroplast phenotype, similar to other Mur knockout lines. The addition of d-Ala-d-Ala (DA-DA) to the medium suppressed the appearance of giant chloroplasts in ∆Pp-ddl, but the addition of l-Ala-l-Ala (LA-LA), DA-LA, LA-DA, or d-Ala did not. Recently, a metabolic method for labeling bacterial peptidoglycan was established using ethynyl-DA-DA (EDA-DA) and click chemistry to attach an azide-modified fluorophore to the ethynyl group. The ∆Pp-ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing d-amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana. more...
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- 2016
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38. Functional and Structural Analyses of the Inactive Recombinant Enzymes from Hyperthermophilic Archaea Produced in Escherichia coli
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Haruhiko Sakuraba, Toshihisa Ohshima, and Shuichiro Goda
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0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Chemistry - Published
- 2016
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39. Catalytic properties and crystal structure of UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase from Phytophthora infestans
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Kazunari Yoneda, Kenji Fukui, Rina Nagano, Tomohiro Araki, Haruhiko Sakuraba, Takuya Mikami, and Toshihisa Ohshima
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Models, Molecular ,Threonine ,Phytophthora infestans ,Glycine ,Bioengineering ,Crystallography, X-Ray ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Biochemistry ,Catalysis ,UDPglucose 4-Epimerase ,Catalytic Domain ,medicine ,Molecular replacement ,Enzyme Inhibitors ,KEGG ,Escherichia coli ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Temperature ,Active site ,Substrate (chemistry) ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Molecular Docking Simulation ,carbohydrates (lipids) ,Alcohol Oxidoreductases ,Enzyme ,biology.protein ,NAD+ kinase ,Biotechnology - Abstract
We report, for the first time, the three-dimensional structure and biochemical properties of a UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase (GalE-like L-ThrDH) from Phytophthora infestans, a plant disease-causing fungus. We identified GalE-like L-ThrDH using Kyoto Encyclopedia of Genes and Genomes (KEGG) database as a candidate target for the development of a new fungicide. The GalE-like L-ThrDH gene was expressed in Escherichia coli, and its product was purified and characterized. N-Acetylglycine was found to act as a competitive inhibitor of the enzyme (Ki =0.18 mM). The crystal structure of the unique hexameric GalE-like L-ThrDH was determined using the molecular replacement method at a resolution of 2.3 A, in the presence of NAD+ and citrate, an analogue of the substrate. Based on the molecular docking simulation, N-acetylglycine molecule was modeled into the active site and the binding mode and inhibition mechanism of N-acetylglycine were elucidated. more...
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- 2020
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40. Effects of multicopper oxidase orientation in multiwalled carbon nanotube biocathodes on direct electron transfer
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Toshihisa Ohshima, Shin-ichiro Suye, Haruhiko Sakuraba, Takenori Satomura, Takuto Nakamura, Hiroaki Sakamoto, and Eiichiro Takamura
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0106 biological sciences ,Nanotube ,Materials science ,Biomedical Engineering ,Bioengineering ,Carbon nanotube ,Multicopper oxidase ,Electrochemistry ,01 natural sciences ,Applied Microbiology and Biotechnology ,law.invention ,Electron Transport ,03 medical and health sciences ,Electron transfer ,Protein Domains ,law ,010608 biotechnology ,Drug Discovery ,Electrodes ,030304 developmental biology ,0303 health sciences ,Nanotubes, Carbon ,Process Chemistry and Technology ,General Medicine ,Enzymes, Immobilized ,Cathode ,Chemical engineering ,Electrode ,Molecular Medicine ,Oxidoreductases ,Current density ,Biotechnology - Abstract
In this study, multicopper oxidase (MCO) was immobilized on multiwalled carbon nanotubes (MWCNTs) at two different orientations, and the electrochemical properties of the resulting cathodes were investigated. Using N- or C-terminal His-tagged MCO and MWCNTs, we constructed two types of cathodes. We assumed that the distance between the type 1 (T1)Cu of the C-terminal His-tagged MCO and the MWCNT surface was lesser than that between the T1Cu of the N-terminal His-tagged MCO and the MWCNT surface. In addition, in the C-terminal His-tagged MCO, T1Cu was expected to be closer to the MWCNT surface than the type 2/type 3 Cu site. The current density of the modified electrode with a C-terminal His-tagged MCO immobilized on an MWCNT surface was 1.3-fold higher than that of the electrode with an N-terminal His-tagged MCO immobilized on an MWCNT surface. In addition, the amount of H2 O2 produced by the N-terminal His-tagged MCO immobilized MWCNT modified electrodes was 2.3-fold higher than that produced by the C-terminal His-tagged MCO immobilized MWCNT electrodes. In direct electron transfer (DET)-type biocathodes, both the MCO orientation and the distance between the T1Cu of MCO and the electrode surface are important. The authors succeeded in constructing highly efficient DET-type electrodes. more...
- Published
- 2018
41. Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application
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Toshihisa Ohshima, Hironaga Akita, Junji Hayashi, and Haruhiko Sakuraba
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0301 basic medicine ,Microbiology (medical) ,D-amino acid ,030106 microbiology ,thermostable D-amino acid dehydrogenase ,lcsh:QR1-502 ,Dehydrogenase ,Review ,D-amino acid dehydrogenase ,crystal structure analysis ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,Ureibacillus thermosphaericus ,meso-diaminopimelate dehydrogenase ,chemistry.chemical_classification ,biology ,Chemistry ,Oxidative deamination ,protein engineering ,Enzyme assay ,Amino acid ,030104 developmental biology ,Enzyme ,stable isotope-labeled D-amino acid ,Biochemistry ,biology.protein ,NAD+ kinase ,Isoleucine - Abstract
Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes. more...
- Published
- 2018
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42. A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3
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Tatsuya Ohshida, Ryushi Kawakami, Haruhiko Sakuraba, and Toshihisa Ohshima
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0301 basic medicine ,Microbiology (medical) ,D-amino acid ,genetic structures ,Stereochemistry ,030106 microbiology ,Mutant ,lcsh:QR1-502 ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Pyrococcus horikoshii ,Ala racemase ,Amino-acid racemase ,PLP-dependent enzyme ,Pyridoxal ,Alanine ,chemistry.chemical_classification ,biology ,Amino-acid racemase activity ,biology.organism_classification ,hyperthermophilic archaea ,Pyrococcus horikoshii OT-3 ,030104 developmental biology ,Enzyme ,chemistry ,Serine racemase ,Ser racemase - Abstract
We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea. more...
- Published
- 2018
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43. Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex
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Kohei Ogata, Sanenori Nakamura, Toshihisa Ohshima, Yui Yajima, Masaru Goto, Ryosuke Kaneko, and Kazuaki Yoshimune
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Models, Molecular ,0301 basic medicine ,Macromolecular Substances ,Stereochemistry ,Molecular Conformation ,Sulfolobus tokodaii ,Homoserine ,lcsh:Medicine ,Ligands ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Oxidoreductase ,Homoserine Dehydrogenase ,Cysteine ,Threonine ,lcsh:Science ,Cysteine metabolism ,Homoserine dehydrogenase ,chemistry.chemical_classification ,Multidisciplinary ,030102 biochemistry & molecular biology ,Chemistry ,Spectrum Analysis ,lcsh:R ,NAD ,030104 developmental biology ,lcsh:Q ,NAD+ kinase ,Protein Binding - Abstract
Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 μM and uncompetitive an inhibitor of NAD and NADP with Ki’s of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD first binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption-difference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring. more...
- Published
- 2018
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44. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology
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Toshihisa Ohshima, Haruhiko Sakuraba, Takenori Satomura, and Shin-ichiro Suye
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D-Amino-Acid Oxidase ,Alanine ,chemistry.chemical_classification ,biology ,Catabolism ,Thermophile ,D-amino acid oxidase ,Biosensing Techniques ,General Medicine ,D-amino acid dehydrogenase ,biology.organism_classification ,Archaea ,Applied Microbiology and Biotechnology ,Substrate Specificity ,Amino acid ,Solubility ,Biochemistry ,chemistry ,Gram-Negative Bacteria ,Amino Acid Sequence ,Amino Acids ,Bacteria ,Biotechnology - Abstract
Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors. more...
- Published
- 2015
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45. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by d-amino acids
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Haruhiko Sakuraba, Taketo Ohmori, Ryushi Kawakami, and Toshihisa Ohshima
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chemistry.chemical_classification ,Organic Chemistry ,Clinical Biochemistry ,Biology ,biology.organism_classification ,Biochemistry ,Amino acid ,Pyrococcus horikoshii ,chemistry.chemical_compound ,Open reading frame ,Enzyme ,chemistry ,Genome, Archaeal ,Amino Acids ,Amino-acid racemase ,Gene ,Pyridoxal ,Amino acid synthesis ,Amino Acid Isomerases - Abstract
To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth. more...
- Published
- 2015
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46. Lactic Fermentation and D-Amino Acid Production
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Yuta MUTAGUCHI, Taketo OHMORI, and Toshihisa OHSHIMA
- Published
- 2015
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47. d-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum
- Author
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Haruhiko Sakuraba, Yoshihiro Takaki, Toshihisa Ohshima, Takenori Satomura, Takuro Nunoura, Shin-ichiro Suye, Ken Takai, Hiroaki Sakamoto, Junji Hayashi, and Hideto Takami
- Subjects
0301 basic medicine ,Hot Temperature ,Archaeal Proteins ,Bioengineering ,Applied Microbiology and Biotechnology ,Biosensing Techniques ,medicine.disease_cause ,Electrochemistry ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Lactate dehydrogenase ,Enzyme Stability ,medicine ,Escherichia coli ,Lactic Acid ,Incubation ,Electrodes ,Lactate Dehydrogenases ,Chromatography ,Thermophile ,010401 analytical chemistry ,Enzymes, Immobilized ,Archaea ,0104 chemical sciences ,030104 developmental biology ,chemistry ,Electrode ,D-lactate dehydrogenase ,Biotechnology - Abstract
A stable d -lactate electrochemical sensing system was developed using a dye-linked d -lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d -lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d -lactate. The electrode response to d -lactate was linear within the concentration range of 0.03–2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d -lactate sensing system using a thermostable Dye-DLDH. more...
- Published
- 2017
48. Crystal structure of the NADP
- Author
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Kazunari, Yoneda, Haruhiko, Sakuraba, Tomohiro, Araki, and Toshihisa, Ohshima
- Subjects
Molecular Docking Simulation ,Alcohol Oxidoreductases ,Hot Temperature ,Archaeal Proteins ,Catalytic Domain ,Enzyme Stability ,Pyrobaculum ,Serine ,Crystallography, X-Ray ,Tartrates ,NADP ,Protein Binding ,Substrate Specificity - Abstract
A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP more...
- Published
- 2017
49. Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase
- Author
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Masahiro Watanabe, Toshihisa Ohshima, Junji Hayashi, Tomonari Seto, Haruhiko Sakuraba, Hironaga Akita, Tamotsu Hoshino, and Kazunari Yoneda
- Subjects
0301 basic medicine ,Models, Molecular ,030106 microbiology ,Mutant ,Lysine ,Amino Acid Motifs ,Dehydrogenase ,D-amino acid dehydrogenase ,Protein Engineering ,Applied Microbiology and Biotechnology ,Substrate Specificity ,03 medical and health sciences ,Bacterial Proteins ,Oxidoreductase ,Amino Acid Sequence ,Enzymology and Protein Engineering ,chemistry.chemical_classification ,Binding Sites ,Ecology ,Chemistry ,Substrate (chemistry) ,Oxidative deamination ,Kinetics ,030104 developmental biology ,Biochemistry ,Planococcaceae ,Mutagenesis, Site-Directed ,NAD+ kinase ,Amino Acid Oxidoreductases ,NADP ,Food Science ,Biotechnology - Abstract
A stable NADP + -dependent d -amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d -phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d -leucine, d -norleucine, d -methionine, d -isoleucine, and d -tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d -amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d -amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d -amino acids, but all include tedious steps. The use of NAD(P) + -dependent d -amino acid dehydrogenase (DAADH) makes single-step production of d -amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d -amino acids, such as d -leucine and d -isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d -amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. more...
- Published
- 2017
50. Complete Genome Sequence of Thermus thermophilus TMY, Isolated from a Geothermal Power Plant
- Author
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Katsumi Doi, Yuko Nagayoshi, Toshihisa Ohshima, Seiya Ogata, and Yasuhiro Fujino
- Subjects
0301 basic medicine ,Genetics ,Whole genome sequencing ,Geothermal power ,biology ,Strain (chemistry) ,030106 microbiology ,Thermus thermophilus ,biology.organism_classification ,03 medical and health sciences ,Plasmid ,Chromosomal dna ,Prokaryotes ,Molecular Biology ,Gene - Abstract
Thermus thermophilus TMY (JCM 10668) was isolated from silica scale formed at a geothermal power plant in Japan. Here, we report the complete genome sequence for this strain, which contains a chromosomal DNA of 2,121,526 bp with 2,500 predicted genes and a pTMY plasmid of 19,139 bp, with 28 predicted genes. more...
- Published
- 2017
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