Tokushima University / Educator and Researcher Directory --- Ohshima, Toshihisa

Field of Study

○	Enzyme Chemistry, Enzyme Engineering, Genetics Engineering

Subject of Study

○	ゲノム情報に基づく超好熱菌酵素の構造と機能解析, 特異環境微生物の環境適応の分子機構の解明と生物工学的応用, アミノ酸代謝関連酵素の遺伝子工学的機能開発, 酵素の機能創製 (超好熱菌, 低温菌, 特異環境微生物, 酵素の機能デザイン, 酵素の応用)

Book / Paper

Book:

1.	Haruhiko Sakuraba and Toshihisa Ohshima : 酵素ハンドブック第3版(分担)(一島英治ら編), 朝倉書店, Tokyo, (Summary) 酵素の反応，測定法，所在と精製，構造と性質，その他の特記事項について要点を記述し，検索に便利なように記載した．
2.	Haruhiko Sakuraba, Toshihisa Ohshima and Hideaki Tsuge : 超好熱菌由来酵素を創薬に利用:2-デオキシリボース-5-リン酸アルドラーゼ, KYORITSU SHUPPAN.CO.,LTD, Tokyo, Apr. 2007.
3.	Toshihisa Ohshima, Haruhiko Sakuraba and Hideaki Tsuge : バイオセンサー素子としてのアミノ酸脱水素酵素の構造と機能, KYORITSU SHUPPAN.CO.,LTD, Tokyo, Apr. 2007.
4.	Toshihisa Ohshima : 農芸化学の事典(分担), 朝倉書店, Tokyo, Oct. 2003.
5.	Toshihisa Ohshima : 微生物の取扱いと利用·応用技術(分担), 情報機構, Tokyo, May 2003.
6.	Toshihisa Ohshima and Kenji Soda : 30.Stereoselective Biocatalysis: Amino Acid Dehydrogenases and Their Applications., Marcel Dekker.Inc, New York, Jan. 2000.
7.	Toshihisa Ohshima : 生化学辞典第3版(共著)(今堀和友，山川民夫監修), Tokyo Kagaku Dozin C., Ltd., 東京都, Oct. 1998.
8.	Toshihisa Ohshima and 健次左右田 : 酵素のおはなし, Japanese Standards Association, 東京都, Nov. 1997.
9.	Toshihisa Ohshima : 細胞機能と代謝マップ，I．細胞の代謝·物質の動態(共著)(日本生化学会編), Tokyo Kagaku Dozin C., Ltd., 東京都, Sep. 1997.
10.	手島光司 and Toshihisa Ohshima : 衝撃波の微生物細胞におよび生体機能成分への影響, シュプリンガフェアーラーク, Jan. 1995.
11.	Toshihisa Ohshima : 酵素関係部分, 岩波書店, Jan. 1995.
12.	Toshihisa Ohshima and 手島光司 : 細胞および細胞機能成分に対する衝撃波の影響, さんえい出版株式会社, Jan. 1994.
13.	江崎信芳, Toshihisa Ohshima and 左右田健次 : 新しい物質変換系の開発アミノ酸脱水素酵素とその応用, 学会出版センタ-, Jan. 1992.
14.	Toshihisa Ohshima and 左右田健次 : 第1巻タンパク質 Ⅴ.酵素·その他の機能タンパク質, 6 補欠分子族の同定と定量, Tokyo Kagaku Dozin C., Ltd., Jan. 1991.
15.	Toshihisa Ohshima and 左右田健次 : 第1巻タンパク質 Ⅴ.酵素·その他の機能タンパク質, 4.7 酵素的サイクリング, Tokyo Kagaku Dozin C., Ltd., Jan. 1991.
16.	Toshihisa Ohshima and Soda Kenji : (Managing Editor: A. Fiechter), Biochemistry and Biotechnology of Amino Acid Dehydrogenases, springer-verlag(berlinheidelberg), Jan. 1990.
17.	Toshihisa Ohshima and 左右田健次 : 第2分冊水溶性ビタミン, 第5章ナイアシン, Kagaku-Dojin Publishing Company,INC, Jan. 1989.
18.	Toshihisa Ohshima and 左右田健次 : 第5分冊ビタミンと医学, ナイアシン, NAD(P)要求性酵素と疾患, Kagaku-Dojin Publishing Company,INC, Jan. 1989.
19.	Toshihisa Ohshima and 左右田健次 : 第5章補酵素のハイブリッド, KYORITSU SHUPPAN.CO.,LTD, Jan. 1988.
20.	Toshihisa Ohshima and 左右田健次 : 第Ⅰ篇第1章第2節バイオテクノロジ-における分離実験の進め方, サイエンスフォ-ラム, Jan. 1987.
21.	Toshihisa Ohshima and 左右田健次 : 第Ⅲ篇バイオ有用物質への応用, 第2章生体高分子への応用, 第4節酵素, サイエンスフォ-ラム, Jan. 1987.
22.	左右田健次 and Toshihisa Ohshima : アミノ酸脱水素酵素. (日本生化学会編), Tokyo Kagaku Dozin C., Ltd., Jan. 1983.
23.	左右田健次, Toshihisa Ohshima and 江崎信芳 : 同位体効果利用による酵素反応研究法.(日本アイソト-プ協会編), MARUZEN Co.,Ltd., Jan. 1983.
24.	Toshihisa Ohshima and 左右田健次 : 微生物反応と微生物による有用物質生産. 環境技術研究会編, 理工新社, Jan. 1983.

Academic Paper (Judged Full Paper):

1.	Ryushi Kawakami, Chinatsu Kinoshita, Tomoki Kawase, Mikio Sato, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : Characterization of novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis., Bioscience, Biotechnology, and Biochemistry, Vol.85, No.7, 1650-1657, 2021. (Summary) 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 5'-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. (Keyword) PLP / amino acid racemase / substrate specificity / Thermococcus litoralis (Tokushima University Institutional Repository) ● Metadata: 116135 (Link to Publication Site) ● Publication site (DOI): 10.1093/bbb/zbab078 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 33942867 ● Search Scopus @ Elsevier (PMID): 33942867 ● Search Scopus @ Elsevier (DOI): 10.1093/bbb/zbab078 (Tokushima University Institutional Repository: 116135, DOI: 10.1093/bbb/zbab078, PubMed: 33942867)
2.	Ryushi Kawakami, Tatsuya Ohshida, Haruhiko Sakuraba and Toshihisa Ohshima : A novel PLP-dependent Alanine/Serine racemase from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3, Frontiers in Microbiology, 2018. (Summary) We recently identified and characterized a novel broad substrate specificity amino acidracemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3.Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about45% sequence identity with BAR were present in the P. horikoshii genome. In this study,we detected pyridoxal 50-phosphate (PLP)-dependent amino acid racemase activity inthe 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 orSer. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASRwas highly stable at high temperatures and over a wide range of pHs, though itshexameric structure differed from the dimeric structure of BAR. No activity was detectedin K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) fromLactobacillus buchneri JCM1115, these residues are involved in Schiff base formationand substrate interaction, respectively. Unlike BAR, enhanced ASR activity was notdetected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is thefirst description of a PLP-dependent fold type I ASR in archaea. (Keyword) Ala racemase / Ser racemase / PLP-dependent enzyme / Pyrococcus horikoshii / hyperthermophilic archaea / D-amino acid (Tokushima University Institutional Repository) ● Metadata: 114287 (Link to Publication Site) ● Publication site (DOI): 10.3389/fmicb.2018.01481 (Link to Search Site for Scientific Articles) ● Summary page in Scopus @ Elsevier: 2-s2.0-85049861690 (Tokushima University Institutional Repository: 114287, DOI: 10.3389/fmicb.2018.01481, Elsevier: Scopus)
3.	Ryushi Kawakami, Haruhiko Sakuraba, Ohmori Taketo and Toshihisa Ohshima : First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3 induced by D-amino acids, Journal of Bioscience and Bioengineering, Vol.124, No.1, 23-27, 2017. (Summary) 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 2h in buffer (pH 710), 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 etal., Amino Acids, 47, 15791587, 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. (Keyword) Amino Acid Isomerases / Amino Acid Sequence / Hydrophobic and Hydrophilic Interactions / Pyrococcus horikoshii / Substrate Specificity / Temperature (Tokushima University Institutional Repository) ● Metadata: 111977 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.jbiosc.2017.02.004 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 28343923 ● Summary page in Scopus @ Elsevier: 2-s2.0-85016976917 (Tokushima University Institutional Repository: 111977, DOI: 10.1016/j.jbiosc.2017.02.004, PubMed: 28343923, Elsevier: Scopus)
4.	Tatsuya Ohshida, Junji Hayashi, Takenori Satomura, Ryushi Kawakami, Toshihisa Ohshima and Haruhiko Sakuraba : First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase, Protein Expression and Purification, Vol.126, 62-68, 2016. (Summary) 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. (Tokushima University Institutional Repository) ● Metadata: 111972 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.pep.2016.05.009 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 27215670 ● Summary page in Scopus @ Elsevier: 2-s2.0-84973496063 (Tokushima University Institutional Repository: 111972, DOI: 10.1016/j.pep.2016.05.009, PubMed: 27215670, Elsevier: Scopus)
5.	Ryushi Kawakami, Taketo Ohmori, Haruhiko Sakuraba and Toshihisa Ohshima : Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids, Amino Acids, Vol.47, No.8, 1579-1587, 2015. (Summary) 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. (Link to Publication Site) ● Publication site (DOI): 10.1007/s00726-015-2001-6 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 25963389 ● Summary page in Scopus @ Elsevier: 2-s2.0-84937518494 (DOI: 10.1007/s00726-015-2001-6, PubMed: 25963389, Elsevier: Scopus)
6.	Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis, Bioscience, Biotechnology, and Biochemistry, Vol.78, No.12, 2045-2050, 2014. (Summary) We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition. (Keyword) Amino Acid Sequence / Bacterial Proteins / Binding Sites / Biocatalysis / Burkholderiaceae / Cysteine / Enzyme Assays / Gene Expression / Glutamate Dehydrogenase / Glutamic Acid / Kinetics / Mercuric Chloride / Molecular Sequence Data / Mutagenesis, Site-Directed / Mutation / Protein Binding / Protein Structure, Tertiary / Protein Subunits / Sequence Homology, Amino Acid / Structure-Activity Relationship (Link to Publication Site) ● Publication site (DOI): 10.1080/09168451.2014.946394 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 25126984 ● Summary page in Scopus @ Elsevier: 2-s2.0-84924529811 (DOI: 10.1080/09168451.2014.946394, PubMed: 25126984, Elsevier: Scopus)
7.	Haruhiko Sakuraba, Kaori Yokono, Kazunari Yoneda, Akira Watanabe, Yasuhiko Asada, Takenori Satomura, Tomoki Yabutani, Junko Motonaka and Toshihisa Ohshima : Catalytic properties and crystal structure of quinoprotein aldose sugar dehydrogenase from hyperthermophilic archaeon Pyrobaculum aerophilum, Archives of Biochemistry and Biophysics, Vol.502, No.2, 81-88, 2010. (Summary) We identified a gene encoding a soluble quinoprotein glucose dehydrogenase homologue in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The enzyme was extremely thermostable, and the activity of the pyrroloquinoline quinone (PQQ)-bound holoenzyme was not lost after incubation at 100 degrees C for 10 min. The crystal structure of the enzyme was determined in both the apoform and as the PQQ-bound holoenzyme. The overall fold of the P. aerophilum enzyme showed significant similarity to that of soluble quinoprotein aldose sugar dehydrogenase (Asd) from E. coli. However, clear topological differences were observed in the two long loops around the PQQ-binding sites of the two enzymes. Structural comparison revealed that the hyperthermostability of the P. aerophilum enzyme is likely attributable to the presence of an extensive aromatic pair network located around a beta-sheet involving N- and C-terminal beta-strands. (Keyword) Archaea / Binding Sites / Carbohydrates / Catalysis / Escherichia coli / Glucose Dehydrogenases / Oxidoreductases / PQQ Cofactor / Pyrobaculum (Link to Publication Site) ● Publication site (DOI): 10.1016/j.abb.2010.08.002 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 20692227 ● Search Scopus @ Elsevier (PMID): 20692227 ● Search Scopus @ Elsevier (DOI): 10.1016/j.abb.2010.08.002 (DOI: 10.1016/j.abb.2010.08.002, PubMed: 20692227)
8.	Kuniko Nagasaka, Hideyuki Nakagawa, Fumihiko Satoh, Takefumi Hosotani, Kumio Yokoigawa, Hitomi Sakai, Haruhiko Sakuraba, Toshihisa Ohshima, Mitsuko Shinohara and Kiyoshi Ohura : A novel cytotoxic protein, Karatoxin, from the dorsal spines of the the redfin velvetfihi, Hypodytes rubripinnis, Toxin Reviews, Vol.28, No.4, 260-265, 2009. (Keyword) Hypodytes rubripinnis / Karatoxin (Link to Publication Site) ● Publication site (DOI): 10.3109/15569540903348940 (Link to Search Site for Scientific Articles) ● Summary page in Scopus @ Elsevier: 2-s2.0-72049118862 (DOI: 10.3109/15569540903348940, Elsevier: Scopus)
9.	Yuji Tani, Yukiko Itoyama, Kenichi Nishi, Chikahiro Wada, Yoshio Shoda, Takenori Satomura, Haruhiko Sakuraba, Toshihisa Ohshima, Yukako Hayashi, Tomoki Yabutani and Junko Motonaka : An amperometric D-amino acid biosensor prepared with a thermostable D-Proline dehydrogenase and a carbon nanotube-ionic liquid gel, Analytical Sciences, Vol.25, No.7, 919-923, 2009. (Summary) Carbon nanotube (CNT) gel, which is composed of a mixture of single-wall CNT, an ionic liquid, and a thermostable D-proline dehydrogenase (D-Pro DH) immobilized electrode was utilized for the determination of D-amino acids (DAAs) in food samples. When a critical comparison with CNT, Ketjen Black (KB), and carbon powder (CP) was also carried out, the CNT/D-Pro DH immobilized electrode showed the highest sensitivity and the lowest detection limit of D-proline. In addition, the CNT/D-Pro DH immobilized electrode was applied to detection of DAAs in rice wine and vinegar samples. The concentrations of DAAs in rice wine and vinegar samples were 0.0210 +/- 0.0001 and 0.55 +/- 0.05 mmol L(-1), respectively. (Keyword) Acetic Acid / Amino Acids / Biosensing Techniques / Electric Conductivity / electrochemistry / Electrodes / Enzyme Stability / Enzymes, Immobilized / Gels / Ionic Liquids / Nanotubes, Carbon / Oryza sativa / Proline Oxidase / surface properties / temperature / Wine (Link to Publication Site) ● Publication site (DOI): 10.2116/analsci.25.919 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 19609033 ● Summary page in Scopus @ Elsevier: 2-s2.0-68249112432 (DOI: 10.2116/analsci.25.919, PubMed: 19609033, Elsevier: Scopus)
10.	Yuji Tani, Katsuhito Tanaka, Tomoki Yabutani, Yuji Mishima, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : Development of a D-amino acids electrochemical sensor based on immobilization of thermostable D-Proline dehydrogenase within agar gel membrane, Analytica Chimica Acta, Vol.619, No.2, 215-220, 2008. (Summary) A novel biosensor for determination of d-amino acids (DAAs) in biological samples by using an electrode based on immobilization of a thermostable d-Proline dehydrogenase (d-Pro DH) within an agar gel membrane was developed. The electrode was simply prepared by spin-coating the agar solution with the d-Pro DH on a glassy carbon (GC) electrode. An electrocatalytic oxidation current of 2,6-dichloroindophenol (DCIP) was observed at -100mV vs. Ag/AgCl with the addition of 5 and 20mmolL(-1)d-proline. The current response and its relative standard deviation were 0.15muA and 7.6% (n=3), respectively, when it was measured in a pH 8.0 phosphate buffer solution containing 10mmolL(-1)d-proline and 0.5mmolL(-1) DCIP at 50 degrees C. The current response of d-proline increased with increase of the temperature of the sample solution up to 70 degrees C. The electrocatalytic response at the d-Pro DH/agar immobilized electrode subsequently maintained for 80 days. Finally, the d-Pro DH/agar immobilized electrode was applied to determination of DAAs in a human urine sample. The determined value of DAAs in the human urine and its R.S.D. were 1.39+/-0.12mmolL(-1) and 8.9% (n=3), respectively. (Keyword) Agar / Amino Acids / Electrochemistry / Electrodes / Enzymes, Immobilized / Gels / Humans / Proline Oxidase / Reproducibility of Results (Link to Publication Site) ● Publication site (DOI): 10.1016/j.aca.2008.04.063 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 18558115 ● CiNii @ National Institute of Informatics (CRID): 1573668926247298688 ● Summary page in Scopus @ Elsevier: 2-s2.0-46549086171 (DOI: 10.1016/j.aca.2008.04.063, PubMed: 18558115, CiNii: 1573668926247298688, Elsevier: Scopus)
11.	Haruhiko Sakuraba, K. Yoneda, I. Asai, Hideaki Tsuge, N Katunuma and Toshihisa Ohshima : Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii., Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1784, No.3, 563-571, 2008. (Summary) The crystal structure of the highly thermostable l-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 A resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an alpha+beta capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a beta-strand that runs parallel to the C-terminal strand. This results in the formation of an additional beta-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO. (Keyword) Amino Acid Oxidoreductases / Amino Acid Sequence / Archaeal Proteins / Bacterial Proteins / Crystallography, X-Ray / Escherichia coli / Flavin-Adenine Dinucleotide / Hot Temperature / Molecular Sequence Data / Protein Conformation / Recombinant Proteins / Sulfolobus (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbapap.2007.12.012 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 18226609 ● Search Scopus @ Elsevier (PMID): 18226609 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbapap.2007.12.012 (DOI: 10.1016/j.bbapap.2007.12.012, PubMed: 18226609)
12.	Yasuhiro Shimizu, Haruhiko Sakuraba, Katsumi Doi and Toshihisa Ohshima : Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii, Archives of Biochemistry and Biophysics, Vol.470, No.2, 120-128, 2007. (Summary) A gene (ST1218) encoding a D-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35kDa and was highly thermostable. It preferably acted as an NAD-dependent D-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon. (Keyword) Enzyme Activation / Molecular Weight / Phosphoglycerate Dehydrogenase / Serine / Signal Transduction / Sulfolobus / Temperature (Link to Publication Site) ● Publication site (DOI): 10.1016/j.abb.2007.11.010 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 18054776 ● Search Scopus @ Elsevier (PMID): 18054776 ● Search Scopus @ Elsevier (DOI): 10.1016/j.abb.2007.11.010 (DOI: 10.1016/j.abb.2007.11.010, PubMed: 18054776)
13.	Haruhiko Sakuraba, Kazunari Yoneda, Kumiko Yoshihara, Kyoko Satoh, Ryushi Kawakami, Yoshihiro Uto, Hideaki Tsuge, Katsuyuki Takahashi, Hitoshi Hori and Toshihisa Ohshima : Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase., Applied and Environmental Microbiology, Vol.73, No.22, 7427-7434, 2007. (Summary) Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs. (Keyword) Acetaldehyde / Aldehyde-Lyases / Archaeal Proteins / Binding Sites / Catalysis / Enzyme Stability / Hot Temperature / Hydrogen-Ion Concentration / Magnetic Resonance Spectroscopy / Models, Molecular / molecular structure / Protein Structure, Secondary / Protein Structure, Tertiary / Pyrobaculum / Ribosemonophosphates / structureactivity relationship / Substrate Specificity / Thermotoga maritima (Link to Publication Site) ● Publication site (DOI): 10.1128/AEM.01101-07 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17905878 ● Search Scopus @ Elsevier (PMID): 17905878 ● Search Scopus @ Elsevier (DOI): 10.1128/AEM.01101-07 (DOI: 10.1128/AEM.01101-07, PubMed: 17905878)
14.	Kazunari Yoneda, Haruhiko Sakuraba, Hideaki Tsuge, Nobuhiko Katunuma and Toshihisa Ohshima : Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase-NAD-citrate ternary complex, The FEBS Journal, Vol.274, No.16, 4315-4325, 2007. (Summary) The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (R(free) = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidus L-aspDH/NAD/citrate ternary complex and the Thermotoga maritima L-aspDH/NAD binary complex showed that A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritima L-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidus L-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two L-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to L-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic L-aspDH. (Keyword) Amino Acid Oxidoreductases / Amino Acid Sequence / Archaeal Proteins / Archaeoglobus fulgidus / Aspartic Acid / Binding Sites / Citric Acid / Crystallography, X-Ray / Enzyme Stability / Hydrogen Bonding / Models, Molecular / Molecular Sequence Data / NAD / Protein Binding / Protein Structure, Tertiary / Sequence Homology, Amino Acid / Substrate Specificity / Thermodynamics (Link to Publication Site) ● Publication site (DOI): 10.1111/j.1742-4658.2007.05961.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17651440 ● Search Scopus @ Elsevier (PMID): 17651440 ● Search Scopus @ Elsevier (DOI): 10.1111/j.1742-4658.2007.05961.x (DOI: 10.1111/j.1742-4658.2007.05961.x, PubMed: 17651440)
15.	Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : Gene cloning and characterization of the very large NAD-dependent L-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil, Journal of Bacteriology, Vol.189, No.15, 5626-5633, 2007. (Summary) NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. (Keyword) Amino Acid Sequence / Bacterial Proteins / Base Sequence / Binding Sites / Catalytic Domain / Chromatography, Gel / Cloning, Molecular / Conserved Sequence / DNA, Bacterial / Electrophoresis, Polyacrylamide Gel / Glutamate Dehydrogenase / Glutamic Acid / Molecular Sequence Data / Molecular Weight / NAD / NADP / Oxalobacteraceae / Protein Subunits / Pseudomonas aeruginosa / Sequence Analysis, DNA / Sequence Homology, Amino Acid / Soil Microbiology / Streptomyces / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1128/JB.00496-07 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17526698 ● Search Scopus @ Elsevier (PMID): 17526698 ● Search Scopus @ Elsevier (DOI): 10.1128/JB.00496-07 (DOI: 10.1128/JB.00496-07, PubMed: 17526698)
16.	Toshihisa Ohshima, Ryushi Kawakami, Yuichiro Kanai, Shuichiro Goda and Haruhiko Sakuraba : Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii., Protein Expression and Purification, Vol.54, No.1, 73-78, 2007. (Summary) 2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways. (Keyword) Archaeal Proteins / Cations, Divalent / Cloning, Molecular / Escherichia coli / glucose / Hydrogen-Ion Concentration / Metals / phosphorylation / Phosphotransferases (Alcohol Group Acceptor) / Purine Nucleotides / Sulfolobus (Link to Publication Site) ● Publication site (DOI): 10.1016/j.pep.2007.02.013 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17407821 ● Search Scopus @ Elsevier (PMID): 17407821 ● Search Scopus @ Elsevier (DOI): 10.1016/j.pep.2007.02.013 (DOI: 10.1016/j.pep.2007.02.013, PubMed: 17407821)
17.	Fumihiko Yamaguchi, Toshihisa Ohshima and Haruhiko Sakuraba : An enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate using NAD synthetase, Analytical Biochemistry: Methods in the Biological Sciences, Vol.364, No.2, 97-103, 2007. (Summary) Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer. (Keyword) Alkaline Phosphatase / Amide Synthases / Animals / Automation / Biological Assay / Evaluation Studies as Topic / Glucose 1-Dehydrogenase / Humans / Hydrogen-Ion Concentration / Models, Chemical / NAD(P)H Dehydrogenase (Quinone) / NAD+ Nucleosidase / NADP / Sensitivity and Specificity / Time Factors (Link to Publication Site) ● Publication site (DOI): 10.1016/j.ab.2007.02.011 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17395143 ● Search Scopus @ Elsevier (PMID): 17395143 ● Search Scopus @ Elsevier (DOI): 10.1016/j.ab.2007.02.011 (DOI: 10.1016/j.ab.2007.02.011, PubMed: 17395143)
18.	Takao YOSHIDA, Ryo IIZUKA, Keisuke ITAMI, Takuo YASUNAGA, Haruhiko Sakuraba, Toshihisa Ohshima, Masafumi YOHDA and Tadashi MARUYAMA : Comparative analysis of the protein folding activities of two chaperonin subunits of Thermococcus strain KS-1; the effects of beryllium fluoride, Extremophiles, Vol.11, No.2, 225-235, 2007. (Summary) We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the alpha- and beta-subunit homooligomers (alpha16mer or beta16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both alpha16mer and beta16mer with equal efficiency. This indicated that BeFx replaces the gamma-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin-ADP-BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin-ADP-BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of alpha16mer and beta16mer are likely similar although there were some apparent differences. While beta16mer-mediated GFP refolding in the presence of ATP-BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP-PNP, the folding mediated by the alpha16mer that proceeded with much lower yields. A mutant of alpha16mer, trapalpha, which traps the unfolded and partially folded substrate protein, did not affect the ATP-BeFx-dependent GFP folding by beta16mer but it suppressed that mediated by alpha16mer to the level of spontaneous folding. These results suggested that beta16mer differed from the alpha16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis. (Keyword) Adenosine Diphosphate / Adenosine Triphosphate / Archaeal Proteins / Beryllium / Chaperonins / Fluorides / Green Fluorescent Proteins / Protein Folding / Thermococcus (Link to Publication Site) ● Publication site (DOI): 10.1007/s00792-006-0026-1 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17072688 ● Search Scopus @ Elsevier (PMID): 17072688 ● Search Scopus @ Elsevier (DOI): 10.1007/s00792-006-0026-1 (DOI: 10.1007/s00792-006-0026-1, PubMed: 17072688)
19.	Nakayama Hitoshi, Shimamura Tatsuro, Imagawa Takahito, Shirai Nobuaki, Ito Takashi, Sako Yoshihiko, Miyano Masashi, Haruhiko Sakuraba, Toshihisa Ohshima, Nomura Norimichi and Tsuge Hideaki : Structure of a hyperthermophilic archaeal homing endonuclease, I-Tsp061I: contribution of cross-domain polar networks to thermostability, Journal of Molecular Biology, Vol.365, No.2, 362-378, 2007. (Summary) A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 A resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed alpha-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I. (Keyword) Amino Acid Motifs / Amino Acid Sequence / Archaeal Proteins / Binding Sites / Crystallization / Crystallography, X-Ray / Endonucleases / Enzyme Stability / Molecular Sequence Data / Protein Structure, Tertiary / Sequence Homology, Amino Acid / Structure-Activity Relationship / Thermoproteus (Link to Publication Site) ● Publication site (DOI): 10.1016/j.jmb.2006.09.066 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17069851 ● CiNii @ National Institute of Informatics (CRID): 1572261551376329472 ● Search Scopus @ Elsevier (PMID): 17069851 ● Search Scopus @ Elsevier (DOI): 10.1016/j.jmb.2006.09.066 (DOI: 10.1016/j.jmb.2006.09.066, PubMed: 17069851, CiNii: 1572261551376329472)
20.	Yoneda Kazunari, Ryushi Kawakami, Tagashira Yuya, Haruhiko Sakuraba, Shuichiro Goda and Toshihisa Ohshima : The first archaeal l-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1764, No.6, 1087-1093, 2006. (Summary) A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 degrees C. The optimum temperature for L-aspartate dehydrogenation was about 80 degrees C. The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal L-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species. (Keyword) Amino Acid Oxidoreductases / Amino Acid Sequence / Archaeoglobus fulgidus / Cloning, Molecular / Dimerization / Enzyme Stability / Escherichia coli / Kinetics / Molecular Sequence Data / Oxaloacetate / Protein Structure, Tertiary / Sequence Homology, Amino Acid (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbapap.2006.04.006 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16731057 ● Search Scopus @ Elsevier (PMID): 16731057 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbapap.2006.04.006 (DOI: 10.1016/j.bbapap.2006.04.006, PubMed: 16731057)
21.	Zheng Haitao, Hirose Yutaka, Kimura Tomokazu, Suye Shin-ichiro, Hori Teruo, Katayama Hideo, Arai3 Jun-ichiro, Ryushi Kawakami and Toshihisa Ohshima : L-Proline sensor based on layer-by-layer immobilization of thermostable dye-linked L-proline dehydrogenase and polymerized mediator, Science and Technology of Advanced Materials, Vol.7, No.3, 243-248, 2006. (Summary) L-Proline sensor has been fabricated through multilayer assembly of recombinant dye-linked L-proline dehydrogenase (L-proDH) originally from hyperthermophilic archaeon (Therococcus profundus) and polymerized mediator, poly(allylamine) ferrocene (PAA-Fc), on a gold electrode by electrostatic layer-by-layer alternate adsorption method. The characteristic redox peaks of ferrocene groups were obvious exhibited in cyclic voltammograms, and both anodic and cathodic peaks increased with PAA-Fc/L-proDH bilayer number which confirmed the formation of a multilayer structure. Further experiments showed that an electrode fabricated in this way catalyzed the oxidation of L-proline. It suggested that polymerized mediator could transfer electrons between electrode surface and immobilized L-proDH. Ampeormetric experiments showed that the current response to L-proline increased with PAA-Fc/L-proDH bilayer number. The stability of the sensor was measured, and it kept a 40% relative response after 30 days storage in buffer under 4 °C. (Link to Publication Site) ● Publication site (DOI): 10.1016/j.stam.2005.12.010 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/j.stam.2005.12.010 (DOI: 10.1016/j.stam.2005.12.010)
22.	Shin-ichi Sakasegawa, Hidehiko Ishikawa, Shigeyuki Imamuna, Haruhiko Sakuraba, Shuichiro Goda and Toshihisa Ohshima : Bilirubin oxidase activity of Bacillus subtilis CotA, Applied and Environmental Microbiology, Vol.72, No.1, 972-975, 2006. (Summary) The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein. (Keyword) Amino Acid Sequence / Bacillus subtilis / Bacterial Proteins / Bilirubin / Enzyme Stability / Hot Temperature / Humans / Hydrogen-Ion Concentration / Laccase / Molecular Sequence Data / Oxidoreductases Acting on CH-CH Group Donors / Spores, Bacterial (Link to Publication Site) ● Publication site (DOI): 10.1128/AEM.72.1.972-975.2006 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16391148 ● Summary page in Scopus @ Elsevier: 2-s2.0-33644856537 (DOI: 10.1128/AEM.72.1.972-975.2006, PubMed: 16391148, Elsevier: Scopus)
23.	Shuichiro Goda, Masaki Kojima, Yoshimi Nishikawa, Chizu Kujo, Ryushi Kawakami, Seiki Kuramitsu, Haruhiko Sakuraba, Yuzuru Hiragi and Toshihisa Ohshima : Intersubunit Interaction Induced by Subunit Rearrangement Is Essential for the Catalytic Activity of the Hyperthermophilic Glutamate Dehydrogenase from Pyrobaculum islandicum., Biochemistry, Vol.44, No.46, 15304-15313, 2005. (Summary) The specific activity of recombinant Pyrobaculum islandicum glutamate dehydrogenase (pis-GDH) expressed in Escherichia coli is much lower than that of the native enzyme. However, when the recombinant enzyme is heated at 90 degrees C or exposed to 5 M urea, the activity increases to a level comparable to that of the native enzyme. Small-angle X-ray scattering measurements revealed that the radius of gyration (R(g,z)) of the hexameric recombinant enzyme was reduced to 47 A from 55 A by either heat or urea, and that the final structure of the active enzyme is the same irrespective of the mechanism of activation. Activation was accompanied by a shift in the peaks of the Kratky plot, though the molecular mass of the enzyme was unchanged. The activation-induced decline in R(g,z) followed first-order kinetics, indicating that activation of the enzyme involved a transition between two states, which was confirmed by singular-value decomposition analysis. When the low-resolution structure of the recombinant enzyme was restored using ab initio modeling, we found it to possess no point symmetry, whereas the heat-activated enzyme possessed 32-point symmetry. In addition, a marked increase in the fluorescence emission was observed with addition of ANS to the inactive recombinant enzyme but not the active forms, indicating that upon activation hydrophobic residues on the surface of the recombinant protein moved to the interior. Taken together, these data strongly suggest that subunit rearrangement, i.e., a change in the quaternary structure of the hexameric recombinant pis-GDH, is essential for activation of the enzyme. (Keyword) Calorimetry, Differential Scanning / Cloning, Molecular / Enzyme Activation / Glutamate Dehydrogenase / Hot Temperature / Hydrophobic and Hydrophilic Interactions / Protein Structure, Quaternary / Pyrobaculum / Recombinant Proteins / Urea / X-Ray Diffraction (Link to Publication Site) ● Publication site (DOI): 10.1021/bi050478l (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16285734 ● Search Scopus @ Elsevier (PMID): 16285734 ● Search Scopus @ Elsevier (DOI): 10.1021/bi050478l (DOI: 10.1021/bi050478l, PubMed: 16285734)
24.	Kazunari Yoneda, Haruhiko Sakuraba, Hideaki Tsuge, Nobuhiko Katunuma, Seiki Kuramitsu, Takeshi Kawabata and Toshihisa Ohshima : The first crystal structure of an archaeal helical repeat protein, Acta Crystallographica. Section F, Structural Biology and Crystallization Communications, Vol.F61, No.Pt 7, 636-639, 2005. (Summary) The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 A resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten alpha-helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein. (Keyword) Algorithms / Amino Acid Sequence / Archaea / Archaeal Proteins / Cloning, Molecular / Crystallography, X-Ray / Escherichia coli / Models, Molecular / Molecular Sequence Data / Open Reading Frames / Protein Conformation / Protein Folding / Protein Structure, Secondary / Protein Structure, Tertiary / Sequence Homology, Amino Acid / Sulfolobus (Link to Publication Site) ● Publication site (DOI): 10.1107/S1744309105019263 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16511116 ● Search Scopus @ Elsevier (PMID): 16511116 ● Search Scopus @ Elsevier (DOI): 10.1107/S1744309105019263 (DOI: 10.1107/S1744309105019263, PubMed: 16511116)
25.	Hideaki Tsuge, Ryushi Kawakami, Haruhiko Sakuraba, Hideo Ago, Masashi Miyano, Kenji Aki, Nobuhiko Katunuma and Toshihisa Ohshima : Crystal Structure of a Novel FAD-, FMN-, and ATP-containing L-Proline Dehydrogenase Complex from Pyrococcus horikoshii, The Journal of Biological Chemistry, Vol.280, No.35, 31045-31049, 2005. (Summary) Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (alphabeta)4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 angstroms. The structure of the beta subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the alpha subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the alpha and beta subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the alpha subunit differs from that of the beta subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system. (Keyword) Adenosine Triphosphate / Archaeal Proteins / Crystallography, X-Ray / Flavin Mononucleotide / Flavin-Adenine Dinucleotide / Models, Molecular / Multienzyme Complexes / Proline Oxidase / Protein Structure, Quaternary / Protein Subunits / Pyrococcus horikoshii (Link to Publication Site) ● Publication site (DOI): 10.1074/jbc.C500234200 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16027125 ● Search Scopus @ Elsevier (PMID): 16027125 ● Search Scopus @ Elsevier (DOI): 10.1074/jbc.C500234200 (DOI: 10.1074/jbc.C500234200, PubMed: 16027125)
26.	Yasuhiro Shimizu, Haruhiko Sakuraba, Ryushi Kawakami, Shuichiro Goda, Yutaka Kawarabayasi and Toshihisa Ohshima : L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, --- gene cloning and enzymatic characterization ---, Extremophiles, Vol.9, No.4, 317-324, 2005. (Summary) A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100 degrees C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL-threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn(2+), Mn(2+) and Co(2+) for the activity, and contained one zinc ion/subunit. The K(m) values for L-threonine and NAD at 50 degrees C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain. (Keyword) Alcohol Oxidoreductases / Amino Acid Sequence / Animals / Catalysis / Cloning, Molecular / Hydrogen / Magnetic Resonance Spectroscopy / Molecular Sequence Data / NADP / Pyrococcus horikoshii / Recombinant Proteins / Sequence Alignment / Sequence Homology, Amino Acid / Serine / Stereoisomerism / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1007/s00792-005-0447-2 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15902509 ● CiNii @ National Institute of Informatics (CRID): 1571417126449425536 ● Search Scopus @ Elsevier (PMID): 15902509 ● Search Scopus @ Elsevier (DOI): 10.1007/s00792-005-0447-2 (DOI: 10.1007/s00792-005-0447-2, PubMed: 15902509, CiNii: 1571417126449425536)
27.	Ryushi Kawakami, Haruhiko Sakuraba, Hideaki Tsuge, Shuichiro Goda, Nobuhiko Katunuma and Toshihisa Ohshima : A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3, The FEBS Journal, Vol.272, No.16, 4044-4054, 2005. (Summary) Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii. (Keyword) Amino Acid Sequence / Chromatography, High Pressure Liquid / Coloring Agents / Genes, Archaeal / Molecular Sequence Data / Proline Oxidase / Pyrococcus horikoshii / Recombinant Proteins / Sequence Homology, Amino Acid (Link to Publication Site) ● Publication site (DOI): 10.1111/j.1742-4658.2005.04810.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16098188 ● Search Scopus @ Elsevier (PMID): 16098188 ● Search Scopus @ Elsevier (DOI): 10.1111/j.1742-4658.2005.04810.x (DOI: 10.1111/j.1742-4658.2005.04810.x, PubMed: 16098188)
28.	Haruhiko Sakuraba, Ryushi Kawakami and Toshihisa Ohshima : First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon Pyrococcus horikoshii, --- Cloning, Expression, and Characterization ---, Applied and Environmental Microbiology, Vol.71, No.8, 4352-4358, 2005. (Summary) The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon. (Keyword) Adenosine Triphosphate / Amino Acid Sequence / Cloning, Molecular / Enzyme Stability / Escherichia coli / Hot Temperature / Hydrogen-Ion Concentration / Kinetics / Molecular Sequence Data / Phosphates / Phosphotransferases (Alcohol Group Acceptor) / Polyphosphates / Pyrococcus horikoshii / Sequence Alignment / Temperature (Link to Publication Site) ● Publication site (DOI): 10.1128/AEM.71.8.4352-4358.2005 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16085824 ● Search Scopus @ Elsevier (PMID): 16085824 ● Search Scopus @ Elsevier (DOI): 10.1128/AEM.71.8.4352-4358.2005 (DOI: 10.1128/AEM.71.8.4352-4358.2005, PubMed: 16085824)
29.	Haruhiko Sakuraba, Hideaki Tsuge, Kazunari Yoneda, Nobuhiko Katunuma and Toshihisa Ohshima : Crystal Structure of the NAD Biosynthetic Enzyme Quinolinate Synthase, The Journal of Biological Chemistry, Vol.280, No.29, 26645-26648, 2005. (Summary) A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. The gene was overexpressed in Escherichia coli, and the crystal structure of the produced enzyme was determined to 2.0 A resolution in the presence of malate, a substrate analogue. The overall structure exhibits a unique triangular architecture composed of a 3-fold repeat of three-layer (alphabetaalpha) sandwich folding. Although some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. The three analogous domains are related to a pseudo-3-fold symmetry. The active site is located at the interface of the three domains and is centered on the pseudo-3-fold axis. The malate molecule is tightly held near the bottom of the active site cavity. The model of the catalytic state during the first condensation step of the quinolinate synthase reaction indicates that the elimination of inorganic phosphate from dihydroxyacetone phosphate may precede the condensation reaction. (Keyword) Archaeal Proteins / Binding Sites / Catalysis / Cloning, Molecular / Crystallography, X-Ray / Escherichia coli / Malates / Multienzyme Complexes / NAD / Phosphates / Protein Conformation / Pyrococcus horikoshii / Structural Homology, Protein (Link to Publication Site) ● Publication site (DOI): 10.1074/jbc.C500192200 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15937336 ● Search Scopus @ Elsevier (PMID): 15937336 ● Search Scopus @ Elsevier (DOI): 10.1074/jbc.C500192200 (DOI: 10.1074/jbc.C500192200, PubMed: 15937336)
30.	Fumihiko Yamaguchi, Takashi Etoh, Mamoru Takahashi, Hideo Misaki, Haruhiko Sakuraba and Toshihisa Ohshima : A new enzymatic cycling method for ammonia assay using NAD synthetase, Clinica Chimica Acta, Vol.352, No.1-2, 165-173, 2005. (Summary) 好熱菌由来のアンモニア依存性NADキナーゼとグルコースデヒドロゲナーゼおよびジアホラーゼを利用した，新規酵素サイクリングアッセイ法を開発した．従来の方法に比べ，この方法では生体成分内の微量のアンモニアを簡便かつ正確に定量できることを示した． (Keyword) Amide Synthases / Ammonia / Clinical Enzyme Tests / Enzyme Stability / Female / Glucose 1-Dehydrogenase / Humans / Male / NADPH Dehydrogenase / Sensitivity and Specificity (Link to Publication Site) ● Publication site (DOI): 10.1016/j.cccn.2004.09.011 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15653111 ● Search Scopus @ Elsevier (PMID): 15653111 ● Search Scopus @ Elsevier (DOI): 10.1016/j.cccn.2004.09.011 (DOI: 10.1016/j.cccn.2004.09.011, PubMed: 15653111)
31.	Shuichiro Goda, Haruhiko Sakuraba, Yutaka Kawarabayasi and Toshihisa Ohshima : The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii, --- cloning, expression, and characterization ---, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1748, No.1, 110-115, 2005. (Summary) 超好熱古細菌Pyrococcus horikoshiiに古細菌で初めてアグマチナーゼを見出し，遺伝子クローニング，大量発現系の構築，および性質決定を行った．超好熱菌のポリアミン代謝に本酵素が機能することを提唱した． (Keyword) Amino Acid Sequence / Archaeal Proteins / Arginine / Cloning, Molecular / Humans / Hydrogen-Ion Concentration / Ornithine / Phylogeny / Putrescine / Pyrococcus horikoshii / Recombinant Proteins / Sequence Alignment / Temperature / Ureohydrolases (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbapap.2004.12.010 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15752699 ● Search Scopus @ Elsevier (PMID): 15752699 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbapap.2004.12.010 (DOI: 10.1016/j.bbapap.2004.12.010, PubMed: 15752699)
32.	Mohammad Wadud Bhuiya, Haruhiko Sakuraba, Toshihisa Ohshima, Takahito Imagawa, Nobuhiko Katunuma and Hideaki Tsuge : The first crystal structure of hyperthremostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum, Journal of Molecular Biology, Vol.345, No.2, 325-337, 2005. (Summary) 超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のNAD依存性グルタミン酸脱水素酵素について，初めてX線結晶構造解析に成功した．これまでグルタミン酸脱水素酵素の高い熱安定性が，イオンペアネットワークの増加に由来するとされてきた定説に対し，サブユニット間の疎水性相互作用の重要性を指摘した．また，補酵素特異性と立体構造の相関を明らかにした． (Keyword) crystal structure / Pyrobaculum islandicum / hyperthermostable NAD-specific glutamate dehydrogenase / hydrophobic interaction in the intersubunit / coenzyme specificity (Link to Publication Site) ● Publication site (DOI): 10.1016/j.jmb.2004.10.063 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15571725 ● Search Scopus @ Elsevier (PMID): 15571725 ● Search Scopus @ Elsevier (DOI): 10.1016/j.jmb.2004.10.063 (DOI: 10.1016/j.jmb.2004.10.063, PubMed: 15571725)
33.	Takahito Imagawa, Hitoshi Nakayama, Nobuhiko Katunuma, Haruhiko Sakuraba, Toshihisa Ohshima, Takashi Itoh, Yoshihiko Sako, Norimichi Nomura and Hideaki Tsuge : Crystallization and preliminary X-ray diffraction analysis of homing endonuclease I-Tsp061I, Acta Crystallographica. Section D, Biological Crystallography, Vol.60, No.11, 2006-2008, 2004. (Summary) 超好熱菌由来新規ホーミングエンドヌクレアーゼを見出し，その結晶化に初めて成功した．また，X線結晶構造の初期解析を行った． (Keyword) Crystallization / Deoxyribonuclease I / Thermoproteus / X-Ray Diffraction (Link to Publication Site) ● Publication site (DOI): 10.1107/S0907444904020232 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15502310 ● Search Scopus @ Elsevier (PMID): 15502310 ● Search Scopus @ Elsevier (DOI): 10.1107/S0907444904020232 (DOI: 10.1107/S0907444904020232, PubMed: 15502310)
34.	Ryushi Kawakami, Haruhiko Sakuraba, Shintaro Kamohara, Shuichiro Goda, Yutaka Kawarabayasi and Toshihisa Ohshima : Oxidative stress response in an anaerobic hyperthermophilic archaeon, --- presence of a functional peroxiredoxin in Pyrococcus horikoshii ---, The Journal of Biochemistry, Vol.136, No.4, 541-547, 2004. (Summary) 嫌気性超好熱古細菌Pyrococcus horikoshiiに僅かな酸素ストレスを与えると，顕著に誘導されるタンパク質を見出し，これがペルオキシレドキシンであることを明らかにした．嫌気性菌が酸素ストレス応答タンパク質を持っており，これが菌体の酸素耐性に関与する可能性を示した． (Keyword) Amino Acid Sequence / Archaeal Proteins / Blotting, Northern / Cloning, Molecular / Culture Media / Databases, Genetic / Dithiothreitol / Electrons / Electrophoresis, Gel, Two-Dimensional / Electrophoresis, Polyacrylamide Gel / Escherichia coli / Genome / Hot Temperature / Humans / Hydrogen-Ion Concentration / Models, Chemical / Molecular Sequence Data / Oxidative Stress / Oxygen / Peroxidases / Peroxiredoxins / Protein Structure, Tertiary / Pyrococcus horikoshii / RNA / Recombinant Proteins / Sequence Homology, Amino Acid / Time Factors (Link to Publication Site) ● Publication site (DOI): 10.1093/jb/mvh157 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15625325 ● Search Scopus @ Elsevier (PMID): 15625325 ● Search Scopus @ Elsevier (DOI): 10.1093/jb/mvh157 (DOI: 10.1093/jb/mvh157, PubMed: 15625325)
35.	Haruhiko Sakuraba, Ryushi Kawakami, Hajime Takahashi and Toshihisa Ohshima : Novel Archaeal Alanine:Glyoxylate Aminotransferase from Thermococcus litoralis, Journal of Bacteriology, Vol.186, No.16, 5513-5518, 2004. (Summary) 超好熱菌に初めてアラニングリオキシル酸アミノトランスフェラーゼを見出した．一次構造解析により系統樹を作製した結果，既知のアラニングリオキシル酸アミノトランスフェラーゼとは全く異なるグループに属することが判明した．本酵素は，芳香族アミノ酸トランスフェラーゼに近縁であるが，反応はアラニンとグリオキシル酸に特異的であった．本菌が本酵素やグリオキシル酸レダクターゼを含むグリオキシル酸代謝経路を持つ事を提唱した． (Keyword) Amino Acid Sequence / Chromatography / DNA, Archaeal / Electrophoresis, Polyacrylamide Gel / Molecular Sequence Data / Phylogeny / Sequence Analysis, DNA / Sequence Analysis, Protein / Sequence Homology / Thermococcus / Transaminases (Link to Publication Site) ● Publication site (DOI): 10.1128/JB.186.16.5513-5518.2004 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15292154 ● Search Scopus @ Elsevier (PMID): 15292154 ● Search Scopus @ Elsevier (DOI): 10.1128/JB.186.16.5513-5518.2004 (DOI: 10.1128/JB.186.16.5513-5518.2004, PubMed: 15292154)
36.	Mohammad W Bhuiya, Haruhiko Sakuraba, Kazunari Yoneda, Toshihisa Ohshima, Takahito Imagawa, Nobuhiko Katunuma and Hideaki Tsuge : Crystallization and preliminary X-ray diffraction analysis of the hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum, Acta Crystallographica. Section D, Biological Crystallography, Vol.60, No.4, 715-717, 2004. (Summary) 超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のグルタミン酸脱水素酵素について，その結晶化に初めて成功した．また，X線結晶構造の初期解析を行った． (Keyword) HYPERTHERMOPHILIC ARCHAEON / PYROCOCCUS-FURIOSUS / THERMOCOCCUS-LITORALIS / PURIFICATION / ENZYME / ARCHAEBACTERIUM / EXPRESSION / PROFUNDUS / CLONING / ALANINE (Link to Publication Site) ● Publication site (DOI): 10.1107/S0907444904001854 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15039563 ● Search Scopus @ Elsevier (PMID): 15039563 ● Search Scopus @ Elsevier (DOI): 10.1107/S0907444904001854 (DOI: 10.1107/S0907444904001854, PubMed: 15039563)
37.	Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : Gene and primary structures of dye-linked L-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex, Extremophiles, Vol.8, No.2, 99-108, 2004. (Summary) 超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，本酵素がユニークなヘテロテトラマー構造をとること，これらをコードする4種の遺伝子がオペロンを形成することを明らかにした．また，この異種サブユニットの共発現系の構築に成功した．本成果および関連研究により，第5回酵素応用シンポジウム研究奨励賞受賞．平成16年度医科学応用研究財団奨励金受賞．成果は日経バイオテク2004/6/21, pp19-20に掲載された． (Keyword) Amino Acid Sequence / Base Sequence / Coloring Agents / Hot Temperature / Macromolecular Substances / Magnetic Resonance Spectroscopy / Molecular Sequence Data / Molecular Weight / Multigene Family / Proline Oxidase / Protein Conformation / Protein Subunits / Sequence Alignment / Sequence Homology, Amino Acid / Thermococcus (Link to Publication Site) ● Publication site (DOI): 10.1007/s00792-003-0368-x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 15064976 ● Search Scopus @ Elsevier (PMID): 15064976 ● Search Scopus @ Elsevier (DOI): 10.1007/s00792-003-0368-x (DOI: 10.1007/s00792-003-0368-x, PubMed: 15064976)
38.	Akira Ideno, Masahiro Furutani, Tokuro Iwabuchi, T Iida, Yoshitaka Iba, Y Kurosawa, Haruhiko Sakuraba, Toshihisa Ohshima, Yutaka Kawarabayasi and Tadashi Maruyama : Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein, Applied Microbiology and Biotechnology, Vol.64, No.1, 99-105, 2004. (Summary) 超好熱菌由来のロダネースの発現に関して，同じく超好熱菌由来のシャペロンと融合させることにより，大腸菌における組換え体タンパク質の生産性が飛躍的に向上することを明らかにした． (Keyword) Archaeal Proteins / Bacterial Proteins / Cloning, Molecular / Electrophoresis, Polyacrylamide Gel / Enzyme-Linked Immunosorbent Assay / Escherichia coli / Gene Expression / Immunoglobulin Fragments / Inclusion Bodies / Molecular Chaperones / Muramidase / Peptidylprolyl Isomerase / Recombinant Fusion Proteins / Solubility / Tacrolimus Binding Proteins / Thiosulfate Sulfurtransferase (Link to Publication Site) ● Publication site (DOI): 10.1007/s00253-003-1459-4 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 14564491 ● CiNii @ National Institute of Informatics (CRID): 1571980076671141248 ● Search Scopus @ Elsevier (PMID): 14564491 ● Search Scopus @ Elsevier (DOI): 10.1007/s00253-003-1459-4 (DOI: 10.1007/s00253-003-1459-4, PubMed: 14564491, CiNii: 1571980076671141248)
39.	Mojgan Heydari, Toshihisa Ohshima, Naoki Nunoura-Kominato and Haruhiko Sakuraba : Highly stable L-lisine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring, --- characterization, gene cloning and sequensing, and expression ---, Applied and Environmental Microbiology, Vol.70, No.2, 937-942, 2004. (Summary) これまでほとんど研究例の無いリジンデヒドロゲナーゼを好熱菌に初めて見出し，その遺伝子クローニングに成功し，一次構造を明らかにした．好熱菌由来の酵素は非常に安定であり，リジンの定量に有用性が高いことを示した． (Keyword) Amino Acid Oxidoreductases / Cloning, Molecular / Enzyme Stability / Fresh Water / Geobacillus stearothermophilus / Hot Temperature / Japan / Lysine / Molecular Sequence Data / Recombinant Proteins / Sequence Analysis, DNA (Link to Publication Site) ● Publication site (DOI): 10.1128/AEM.70.2.937-942.2004 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 14766574 ● Search Scopus @ Elsevier (PMID): 14766574 ● Search Scopus @ Elsevier (DOI): 10.1128/AEM.70.2.937-942.2004 (DOI: 10.1128/AEM.70.2.937-942.2004, PubMed: 14766574)
40.	Reina Katoh, Shinji Nagata, Akira Ozawa, Toshihisa Ohshima, Masahiro Kamekura and Haruo Misono : Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS-3, Journal of Molecular Catalysis B: Enzymatic, Vol.23, No.2-6, 231-238, 2003. (Keyword) L-leucine dehydrogenase / Natronobactrium magadii / halophilic dehydrogenase / alkaliphilic halophile / L-leucine / GENE CLONING / BACILLUS-STEAROTHERMOPHILUS / AMINO-ACIDS / ELECTROPHORESIS / thermophile / PROTEINS / CEREUS (Link to Publication Site) ● Publication site (DOI): 10.1016/S1381-1177(03)00085-7 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/S1381-1177(03)00085-7 (DOI: 10.1016/S1381-1177(03)00085-7)
41.	Shinji Nagata, Yuki Kobayashi, Sanae Shinkawa, Reina Katoh, Toshihisa Ohshima and Haruo Misono : Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137, Journal of Molecular Catalysis B: Enzymatic, Vol.23, No.2-6, 223-230, 2003. (Link to Publication Site) ● Publication site (DOI): 10.1016/S1381-1177(03)00084-5 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/S1381-1177(03)00084-5 (DOI: 10.1016/S1381-1177(03)00084-5)
42.	Shin-ichiro Kato, Toshihisa Ohshima, Andrey Galkin, Ljudmila Kulakova, Tohru Yoshimura and Nobuyoshi Esaki : Purification and characterization of alanine dehydrogenase from a marine bacterium, Vibro proteolyticus, Journal of Molecular Catalysis B: Enzymatic, Vol.23, No.2-6, 373-378, 2003. (Link to Publication Site) ● Publication site (DOI): 10.1016/S1381-1177(03)00101-2 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/S1381-1177(03)00101-2 (DOI: 10.1016/S1381-1177(03)00101-2)
43.	Toshihisa Ohshima, Yoshihiro Ito, Haruhiko Sakuraba, Shuichiro Goda and Yutaka Kawarabayasi : The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase, Journal of Molecular Catalysis B: Enzymatic, Vol.23, No.2-6, 281-289, 2003. (Summary) 超好熱菌由来のグルコース脱水素酵素の遺伝子クローニング，大量発現系の構築に成功した．本酵素の補酵素について立体特異性を明らかにした． (Keyword) Sulfolobus tokodaii / NAD(P)-dependent glucose-1-dehydrogenase / thermoacidophilic archaeon / modified Entner-Doudoroff pathway / primary structure of glucose dehydrogenase / BACILLUS-MEGATERIUM / PURIFICATION / METABOLISM / SEQUENCE / PROTEINS / ENZYME / PATHWAY (Link to Publication Site) ● Publication site (DOI): 10.1016/S1381-1177(03)00091-2 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/S1381-1177(03)00091-2 (DOI: 10.1016/S1381-1177(03)00091-2)
44.	Haruhiko Sakuraba, Kyouhei Kanai, Shuichiro Goda, Yutaka Kawarabayasi and Toshihisa Ohshima : A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, Journal of Molecular Catalysis B: Enzymatic, Vol.23, No.2-6, 273-279, 2003. (Summary) 超好熱菌由来のニコチンアミドモノヌクレオチドアデニリルトランスフェラーゼが高温環境下では，ATPだけでなくADPやAMPもアデニリルドナーとして利用可能なことを示した．この新規な反応性の応用面での有用性を示した． (Link to Publication Site) ● Publication site (DOI): 10.1016/S1381-1177(03)00090-0 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/S1381-1177(03)00090-0 (DOI: 10.1016/S1381-1177(03)00090-0)
45.	Haruhiko Sakuraba, Hideaki Tsuge, Ikuko Shimoya, Ryushi Kawakami, Shuichiro Goda, Yutaka Kawarabayasi, Nobuhiko Katunuma, Hideo Ago, Masashi Miyano and Toshihisa Ohshima : The First Crystal Structure of Archaeal Aldolase: Unique Tetrameric Structure of 2-Deoxy-D-ribose-5-phosphate Aldolase from Hyperthermophilic Archaeon Aeropyrum pernix, The Journal of Biological Chemistry, Vol.278, No.12, 10799-10806, 2003. (Summary) 古細菌のアルドラーゼに関して，初めてX線結晶構造解析に成功した．解析したデオキシリボース5-リン酸アルドラーゼは抗がん剤や抗ウイルス剤の合成中間体の生産に利用できるが，既知の酵素は不安定で産業への実用化が難しい．本酵素は非常に安定性が高く，実用面での有用性を示した．また，その安定性が本酵素のユニークな4次構造に由来することを提唱した．本研究により，平成15年度バイオインダストリー協会「グラント」奨励金受賞． (Keyword) Amino Acid Sequence / Archaea / Archaeal Proteins / Enzyme Stability / Escherichia coli / Fructose-Bisphosphate Aldolase / Hydrogen-Ion Concentration / Molecular Sequence Data / Protein Subunits / Recombinant Proteins / Ribosemonophosphates (Link to Publication Site) ● Publication site (DOI): 10.1074/jbc.M212449200 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12529358 ● Search Scopus @ Elsevier (PMID): 12529358 ● Search Scopus @ Elsevier (DOI): 10.1074/jbc.M212449200 (DOI: 10.1074/jbc.M212449200, PubMed: 12529358)
46.	Haruhiko Sakuraba, Yuri Mitani, Shuichiro Goda, Yutaka Kawarabayasi and Toshihisa Ohshima : Cloning, Expression, and Characterization of the First Archaeal ATP-Dependent Glucokinase from Aerobic Hyperthermophilic Archaeon, Aeropyrum pernix, The Journal of Biochemistry, Vol.133, No.2, 219-224, 2003. (Summary) 超好熱古細菌由来のATP依存性グルコキナーゼについて，初めて遺伝子クローニング，発現系の構築および産物の性質決定を行った．進化的に最古と言われる超好熱菌の酵素を解析し，グルコキナーゼの進化において重要な知見を与えた． (Keyword) Aeropyrum pernix / glucokinase / hexokinase / hyperthermophile / archaea sugar kinase (Link to Publication Site) ● Publication site (DOI): 10.1093/jb/mvg027 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12761185 ● CiNii @ National Institute of Informatics (CRID): 1390001204931265664 ● Search Scopus @ Elsevier (PMID): 12761185 ● Search Scopus @ Elsevier (DOI): 10.1093/jb/mvg027 (DOI: 10.1093/jb/mvg027, PubMed: 12761185, CiNii: 1390001204931265664)
47.	Fumihiko Yamaguchi, Shinji Koga, Issei Yoshioka, Mamoru Takahashi, Haruhiko Sakuraba and Toshihisa Ohshima : Stable Ammonia-specific NAD Synthetase from Bacillus stearothermophilus: Purification, Characterization, Gene Cloning, and Applications, Bioscience, Biotechnology, and Biochemistry, Vol.66, No.10, 2052-2059, 2002. (Summary) Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible. (Keyword) ammonia-specific NAD synthetase / NAD biosynthesis / gene cloning / Bacillus stearothermophilus (Link to Publication Site) ● Publication site (DOI): 10.1271/bbb.66.2052 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12450114 ● CiNii @ National Institute of Informatics (CRID): 1390282681452255872 ● Summary page in Scopus @ Elsevier: 2-s2.0-1842848056 (DOI: 10.1271/bbb.66.2052, PubMed: 12450114, CiNii: 1390282681452255872, Elsevier: Scopus)
48.	Hideaki Tsuge, Haruhiko Sakuraba, Toru Kobe, Akira Kujime, Nobuhiko Katunuma and Toshihisa Ohshima : Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-Å resolution: A large conformational change in ADP-dependent glucokinase, Protein Science, Vol.11, No.10, 2456-2463, 2002. (Summary) Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor. Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway. Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme. Recently, the three-dimensional structure of the ADPGK from T. litoralis (tlGK) in a complex with ADP was reported. The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large alpha/beta-domains and small domains. However, a marked adjustment of the two domains, which is a 10-A translation and a 20 degrees rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo-phGK and ADP-tlGK structures. The ADP-binding loop (430-439) was disordered in the apo form. It is suggested that a large conformational change takes place during the enzymatic reaction. (Keyword) Amino Acid Sequence / Crystallography, X-Ray / Glucokinase / Molecular Sequence Data / Protein Conformation / Pyrococcus / Sequence Alignment / Thermococcus (Link to Publication Site) ● Publication site (DOI): 10.1110/ps.0215602 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12237466 ● Summary page in Scopus @ Elsevier: 2-s2.0-0036785588 (DOI: 10.1110/ps.0215602, PubMed: 12237466, Elsevier: Scopus)
49.	Mohammad W Bhuiya, Hideaki Tsuge, Haruhiko Sakuraba, Kazunari Yoneda, Nobuhiko Katunuma and Toshihisa Ohshima : Crystallization and preliminary X-ray diffraction analysis of glutamate dehydrogenase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1, Acta Crystallographica. Section D, Biological Crystallography, Vol.58, No.8, 1338-1339, 2002. (Summary) 超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のグルタミン酸脱水素酵素の結晶化に初めて成功した．X線結晶構造の初期解析を行った． (Keyword) PYROCOCCUS-FURIOSUS / PYROBACULUM-ISLANDICUM / THERMOCOCCUS-LITORALIS / ENZYME / PURIFICATION / TEMPERATURES / PROFUNDUS / ALANINE / GENE (Link to Publication Site) ● Publication site (DOI): 10.1107/S0907444902008478 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12136148 ● Search Scopus @ Elsevier (PMID): 12136148 ● Search Scopus @ Elsevier (DOI): 10.1107/S0907444902008478 (DOI: 10.1107/S0907444902008478, PubMed: 12136148)
50.	Tatyana A Muranova, Segey N Ruzheinikova, E S Sedelnikova, Patrick J Baker, Alessandra Pasquo, Andrey Galkin, Nobuyoshi Esaki, Toshihisa Ohshima, Kenji Soda and David W Rice : Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermedius, Acta Crystallographica. Section D, Biological Crystallography, Vol.58, No.Pt 6 Pt 2, 1059-1062, 2002. (Summary) Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD(+). Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an 'open' and 'closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD(+) have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 106, b = 118, c = 320 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.1 A(3) Da(-1). The crystals of the non-productive ternary complex belong to space group P6(1) or P6(5), with approximate unit-cell parameters a = b = 117, c = 502 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.0 A(3) Da(-1). These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3 A resolution, respectively, and are suitable for a full structure determination. (Keyword) Amino Acid Oxidoreductases / crystallization / X-ray crystallography / Leucine Dehydrogenase / Micromonosporaceae / Protein Conformation / Recombinant Proteins / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1107/S0907444902006431 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12037317 ● Search Scopus @ Elsevier (PMID): 12037317 ● Search Scopus @ Elsevier (DOI): 10.1107/S0907444902006431 (DOI: 10.1107/S0907444902006431, PubMed: 12037317)
51.	Shin-ichi Sakasegawa, Hideki Takehara, Issei Yoshioka, Hideo Misaki, Haruhiko Sakuraba and Toshihisa Ohshima : Stabilization of Flavobacterium meningosepticum Glycerol Kinase by Introduction of a Hydrogen Bond, Bioscience, Biotechnology, and Biochemistry, Vol.66, No.6, 1374-1377, 2002. (Summary) The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed. (Keyword) Amino Acid Substitution / Enzyme Stability / Flavobacterium / Glycerol Kinase / Hydrogen Bonding / Models, Molecular / Protein Conformation / Temperature (Link to Publication Site) ● Publication site (DOI): 10.1271/bbb.66.1374 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12162561 ● CiNii @ National Institute of Informatics (CRID): 1390282681451619712 ● Search Scopus @ Elsevier (PMID): 12162561 ● Search Scopus @ Elsevier (DOI): 10.1271/bbb.66.1374 (DOI: 10.1271/bbb.66.1374, PubMed: 12162561, CiNii: 1390282681451619712)
52.	Haruhiko Sakuraba, Issei Yoshioka, Shinji Koga, Mamoru Takahashi, Yuki Kitahama, Takenori Satomura, Ryushi Kawakami and Toshihisa Ohshima : ADP-Dependent Glucokinase/Phosphofructokinase, a Novel Bifunctional Enzyme from the Hyperthermophilic Archaeon Methanococcus jannaschii, The Journal of Biological Chemistry, Vol.277, No.15, 12495-12498, 2002. (Summary) 超好熱アーキアMethanococcus jannashiiに見出したグルコキナーゼおよびホスホフルクトキナーゼの両活性を持つ新規酵素について報告している．この酵素がグルコキナーゼおよびホスホフルクトキナーゼの起源であることを提唱した．本酵素の発見により，メタン菌による糖からのメタン生産が理論上可能であることを示した．生化学分野のトップジャーナルの1つであるJ. Biol. Chem.において，特に重要な発見を紹介するAccelerated Publicationに掲載された．本研究により平成14年度ビタミン学会奨励賞受賞． (Keyword) Amino Acid Sequence / Base Sequence / Catalysis / DNA Primers / Glucokinase / Methanococcus / Molecular Sequence Data / Phosphorylation / Phosphotransferases (Alcohol Group Acceptor) / Phylogeny / Recombinant Proteins / Sequence Homology, Amino Acid (Link to Publication Site) ● Publication site (DOI): 10.1074/jbc.C200059200 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11856730 ● CiNii @ National Institute of Informatics (CRID): 1360574094658110464 ● Search Scopus @ Elsevier (PMID): 11856730 ● Search Scopus @ Elsevier (DOI): 10.1074/jbc.C200059200 (DOI: 10.1074/jbc.C200059200, PubMed: 11856730, CiNii: 1360574094658110464)
53.	Takenori Satomura, Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : Dye-linked D-Proline Dehydrogenase from Hyperthermophilic Archaeon Pyrobaculum islandicum Is a Novel FAD-dependent Amino Acid Dehydrogenase, The Journal of Biological Chemistry, Vol.277, No.15, 12861-12867, 2002. (Summary) 超好熱アーキアPyrobaculum islandicumに初めて見出した新規色素依存性D-プロリン脱水素酵素に関して報告している．本酵素は従来報告されているD-アミノ酸脱水素酵素とは大きく構造や性質が異なることを明らかにした．D-アミノ酸の合成およびD-プロリンの定量用バイオセンサー素子としての有用性が期待できる． (Keyword) Amino Acid Sequence / Base Sequence / Chromatography, Ion Exchange / Coloring Agents / DNA Probes / Electrophoresis, Polyacrylamide Gel / Enzyme Stability / Molecular Sequence Data / Proline Oxidase / Sequence Homology, Amino Acid / Thermoproteaceae (Link to Publication Site) ● Publication site (DOI): 10.1074/jbc.M112272200 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11823469 ● CiNii @ National Institute of Informatics (CRID): 1364233269761772032 ● Search Scopus @ Elsevier (PMID): 11823469 ● Search Scopus @ Elsevier (DOI): 10.1074/jbc.M112272200 (DOI: 10.1074/jbc.M112272200, PubMed: 11823469, CiNii: 1364233269761772032)
54.	Fumihiko Satoh, Hideyuki Nakagawa, Haruo Yamada, Kuniko Nagasaka, Toshinori Nagasaka, Yasutetsu Araki, Yasuhiro Tomihara, Masatoshi Nozaki, Haruhiko Sakuraba, Toshihisa Ohshima, Tomomitsu Hatakeyama and Haruhiko Aoyagi : Fishing for bioactive substances from scorpionfish and some sea urchins, Journal of Natural Toxins, Vol.11, No.4, 297-304, 2002. (Summary) Venom proteins from the dorsal spine of two scorpionfish, Hypodytes rubripinnis and Synanceia verrucosa were assayed for mitogenicity and cytotoxicity. The two venoms had both mitogenic and cytotoxic activity on murine splenocytes and murine P388 leukemic cells. In H. rubripinnis, the second gel chromatographic fraction showed cytotoxic activity on P388 leukemic cells. On native PAGE, the glycoprotein isolated by concavalin A sepharose chromatography appeared to have a molecular mass of 110 kDa. In addition, two D-galactose-binding lectins (SUL-I and SUL-II) and a heparin-binding lectin (TGL-I) were purified from the globiferous pedicellariae of the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla, respectively. SUL-I (Nakagawa et al., 1999a) had mitogenic activity and cytotoxic activity but SUL-II and TGL-I did not. SUL-I did not show sequence homology to SUL-II. A hemolytic lectin with a molecular mass of 29 kDa was isolated from the coelomic fluid of T. gratilla. The hemolytic activity of the lectin was dependent on Ca2+ concentration and inhibited by lactose. The present results suggest that some species of scorpionfish and sea urchins may be novel sources for biologically active substances such as anti-tumor compounds or new lectins. (Keyword) Sea urchin venom / Fish venom / SUL-I / Toxopneustes pileolus / Hypodytes rubripinnis (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12503872 ● Summary page in Scopus @ Elsevier: 2-s2.0-0036915041 (PubMed: 12503872, Elsevier: Scopus)
55.	Mohammad W Bhuiya, Haruhiko Sakuraba and Toshihisa Ohshima : Temperature Dependence of Kinetic Parameters for Hyperthermophilic Glutamate Dehydrogenase from Aeropyrum pernix K1, Bioscience, Biotechnology, and Biochemistry, Vol.66, No.4, 873-876, 2002. (Summary) The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively. (Keyword) Aeropyrum pernix K1 / steady state kinetics / glutamate dehydrogenase / hyperthermophile / K m (Link to Publication Site) ● Publication site (DOI): 10.1271/bbb.66.873 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12036066 ● CiNii @ National Institute of Informatics (CRID): 1390001206475257600 ● Search Scopus @ Elsevier (PMID): 12036066 ● Search Scopus @ Elsevier (DOI): 10.1271/bbb.66.873 (DOI: 10.1271/bbb.66.873, PubMed: 12036066, CiNii: 1390001206475257600)
56.	Haruhiko Sakuraba, Takenori Satomura, Ryushi Kawakami, Sanae Yamamoto, Yutaka Kawarabayasi, Hisasi Kikuchi and Toshihisa Ohshima : L-Aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing, Extremophiles, Vol.6, No.4, 275-281, 2002. (Summary) 超好熱アーキアPyrococcus horikoshiiに嫌気性菌で初めて見出したL-アスパラギン酸オキシダーゼについて報告している．本酵素が嫌気条件下でも機能する色素依存性L-アスパラギン酸脱水素酵素反応も触媒することを見出し，NAD生合成系の初発酵素として機能していることを提唱した．本酵素に特徴的な脱水素酵素反応によるL-アスパラギン酸定量への応用が期待できる．本研究により平成14年度ビタミン学会奨励賞受賞． (Keyword) Amino Acid Oxidoreductases / Amino Acid Sequence / Cloning, Molecular / Enzyme Stability / Genome, Archaeal / Hydrogen-Ion Concentration / Kinetics / Molecular Sequence Data / NAD / Pyrococcus / Recombinant Proteins / Substrate Specificity / Temperature (Link to Publication Site) ● Publication site (DOI): 10.1007/s00792-001-0254-3 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12215812 ● Search Scopus @ Elsevier (PMID): 12215812 ● Search Scopus @ Elsevier (DOI): 10.1007/s00792-001-0254-3 (DOI: 10.1007/s00792-001-0254-3, PubMed: 12215812)
57.	Haruhiko Sakuraba, Yoshinori Takamatsu, Takenori Satomura, Ryushi Kawakami and Toshihisa Ohshima : Purification, Characterization, and Application of a Novel Dye-Linked L-Proline Dehydrogenase from a Hyperthermophilic Archaeon, Thermococcus profundus, Applied and Environmental Microbiology, Vol.67, No.4, 1470-1475, 2001. (Summary) 超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素について報告している．本酵素は新規色素依存性酵素の中では非常に安定性が高いことを明らかにした．果物やハチミツの品質管理マーカーであるL-プロリンの定量用バイオセンサー素子としての有用性を明らかにした． (Keyword) 2,6-Dichloroindophenol / Amino Acid Sequence / Coloring Agents / Molecular Sequence Data / Proline / Proline Oxidase / Substrate Specificity / Thermococcus (Link to Publication Site) ● Publication site (DOI): 10.1128/AEM.67.4.1470-1475.2001 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11282592 ● Search Scopus @ Elsevier (PMID): 11282592 ● Search Scopus @ Elsevier (DOI): 10.1128/AEM.67.4.1470-1475.2001 (DOI: 10.1128/AEM.67.4.1470-1475.2001, PubMed: 11282592)
58.	Haruhiko Sakuraba, Emi Utsumi, Harold J. Schreier and Toshihisa Ohshima : Transcriptional Regulation of Phosphoenolpyruvate Synthase by Maltose in the Hyperthermophilic Archaeon, Pyrococcus furiosus, Journal of Bioscience and Bioengineering, Vol.92, No.2, 108-113, 2001. (Summary) The phosphoenolpyruvate synthase (EC 2.7.9.2) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the Pi-dependent formation of pyruvate, ATP and water from phosphoenolpyruvate and AMP [Sakuraba, H., Arch. Biochem. Biophys., 364, 125-128 (1999)]. In this study, the P. furiosus phosphoenolpyruvate synthase was purified to homogeneity and the N-terminal amino acid sequence was determined to be AYRFIKWFEELS. The sequence coincided completely with the N-terminal amino acid sequence of the translation product of the mlrA gene that was found to be upregulated at the transcriptional level by the alpha-linked glucose disaccharide maltose [Robinson, K.A. and Schreier, H.J., Gene, 151, 173-176 (1994)]. The mlrA gene was cloned and expressed in Escherichia coli. The recombinant cells produced a hyperthermostable phosphoenolpyruvate synthase. This indicates that the mlrA gene encodes the enzyme. When P. furiosus was grown on the medium supplemented with maltose, the specific activity of the enzyme markedly increased (about 10-fold) compared with that produced by the cells grown on the medium without maltose. Northern blot analysis revealed enhanced transcription of the mlrA gene in the presence of maltose. These results indicate that transcriptional regulation of phosphoenolpyruvate synthase by maltose is present in P. furiosus. (Link to Publication Site) ● Publication site (DOI): 10.1016/S1389-1723(01)80209-7 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16233068 ● Summary page in Scopus @ Elsevier: 2-s2.0-0034854830 (DOI: 10.1016/S1389-1723(01)80209-7, PubMed: 16233068, Elsevier: Scopus)
59.	Shin-ichi Sakasegawa, Hideki Takehara, Issei Yoshioka, Mamoru Takahashi, Yoshitaka Kagimoto, Hideo Misaki, Haruhiko Sakuraba and Toshihisa Ohshima : Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region, Protein Engineering, Vol.14, No.9, 663-667, 2001. (Summary) 高脂血症診断用試薬として多用されるグリセロールキナーゼに対し1アミノ酸残基置換を行うだけでその安定性を高めた成果を報告している．導入部位は酵素の活性部位から離れたところにあり，酵素活性に影響を及ばさず安定性の向上に成功した．他の酵素にもこの手法の応用が期待できる． (Keyword) adenosine triphosphate / Amino Acid Sequence / Amino Acid Substitution / Aspartic Acid / Base Sequence / Binding Sites / Enzyme Stability / Flavobacterium / Gene Expression Regulation, Bacterial / Glycerol Kinase / Hot Temperature / Models, Molecular / Mutagenesis / Protein Structure, Tertiary / Recombinant Proteins / Sequence Alignment / Serine / Substrate Specificity / Time Factors (Link to Publication Site) ● Publication site (DOI): 10.1093/protein/14.9.663 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11707612 ● Search Scopus @ Elsevier (PMID): 11707612 ● Search Scopus @ Elsevier (DOI): 10.1093/protein/14.9.663 (DOI: 10.1093/protein/14.9.663, PubMed: 11707612)
60.	Toshihisa Ohshima, Naoki Nunoura, Tomomi Kudome and Haruhiko Sakuraba : A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis, --- Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli ---, European Journal of Biochemistry, Vol.268, No.17, 4740-4747, 2001. (Summary) A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed. (Keyword) Alcohol Oxidoreductases / Amino Acid Sequence / Base Sequence / Cloning, Molecular / Coenzymes / DNA, Archaeal / Escherichia coli / Genomic Library / Molecular Sequence Data / Recombinant Proteins / Sequence Homology, Amino Acid / Substrate Specificity / Thermococcus (Link to Publication Site) ● Publication site (DOI): 10.1046/j.1432-1327.2001.02394.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11532010 ● Summary page in Scopus @ Elsevier: 2-s2.0-0035725687 (DOI: 10.1046/j.1432-1327.2001.02394.x, PubMed: 11532010, Elsevier: Scopus)
61.	Mohammad W Bhuiya, Haruhiko Sakuraba, Chizu Kujo, Naoki Nunoura, Yutaka Kawarabayasi, Hisasi Kikuchi and Toshihisa Ohshima : Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene and phylogenetic implications., Extremophiles, Vol.4, No.6, 333-341, 2000. (Summary) NADP-dependent glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95 degrees C for 30 min, and the maximum activity in L-glutamate deamination was obtained around 100 degrees C. The enzyme showed a strict specificity for L-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The Km values for NADP, L-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms. (Keyword) Amino Acid Sequence / Cloning, Molecular / Desulfurococcaceae / Enzyme Stability / Glutamate Dehydrogenase / Hydrogen-Ion Concentration / Kinetics / Molecular Sequence Data / Molecular Weight / Phylogeny / Recombinant Proteins / Sequence Homology, Amino Acid / Substrate Specificity / Temperature (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11139075 ● Summary page in Scopus @ Elsevier: 2-s2.0-0034572477 (PubMed: 11139075, Elsevier: Scopus)
62.	Shinji Koga, Issei Yoshioka, Haruhiko Sakuraba, Mamoru Takahashi, Shinichi Sakasegawa, Sakayu Shimizu and Toshihisa Ohshima : Biochemical Characterization, Cloning, and Sequencing of ADP-Dependent (AMP-Forming) Glucokinase from Two Hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis., The Journal of Biochemistry, Vol.128, No.6, 1079-1086, 2000. (Summary) The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed. (Keyword) Amino Acid Sequence / Base Sequence / Cloning, Molecular / DNA, Archaeal / Glucokinase / Molecular Sequence Data / Pyrococcus furiosus / Sequence Homology, Amino Acid / Substrate Specificity / Thermococcus (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11098152 ● Summary page in Scopus @ Elsevier: 2-s2.0-0034529961 (PubMed: 11098152, Elsevier: Scopus)
63.	Maria Spies, Yuri Kil, Ryoji Masui, Ryuichi Kato, Chizu Kujo, Toshihisa Ohshima, Seiki Kuramitsu and Vladislav Lanzov : The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75℃, European Journal of Biochemistry, Vol.267, No.4, 1125-1137, 2000. (Summary) The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C. It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature. (Keyword) Adenosine Triphosphatases / Adenosine Triphosphate / Amino Acid Sequence / Archaeal Proteins / Catalysis / Cations, Divalent / Cloning, Molecular / DNA / DNA, Single-Stranded / DNA-Binding Proteins / Enzyme Activation / Enzyme Stability / Escherichia coli / Kinetics / Molecular Sequence Data / Protein Structure, Secondary / Rec A Recombinases / Recombinant Proteins / Sequence Alignment / Temperature / Thermodynamics / Thermoproteaceae (Link to Publication Site) ● Publication site (DOI): 10.1046/j.1432-1327.2000.01108.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10672022 ● Summary page in Scopus @ Elsevier: 2-s2.0-0033952524 (DOI: 10.1046/j.1432-1327.2000.01108.x, PubMed: 10672022, Elsevier: Scopus)
64.	Haruhiko Sakuraba, Emi Utsumi, Chizu Kujo and Toshihisa Ohshima : An AMP-Dependent(ATP-Forming) Kinase in the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization and Novel Physiological Role., Archives of Biochemistry and Biophysics, Vol.364, No.1, 125-128, 1999. (Summary) 超好熱古細菌 Pyrococcus furiosus にAMPからATPを合成する新規キナーゼが存在することを初めて見出し，これを単離精製し，その酵素化学的諸性質を明らかにした．また，これまで不明であった超好熱古細菌のエネルギー代謝における本酵素の新しい役割を提唱した． (Keyword) Adenosine Diphosphate / Adenosine Triphosphate / Bacterial Proteins / Chromatography, Gel / Chromatography, High Pressure Liquid / Chromatography, Ion Exchange / Electrophoresis, Polyacrylamide Gel / Hydrogen-Ion Concentration / Molecular Weight / Phosphoenolpyruvate / Phosphotransferases (Phosphate Group Acceptor) / Pyrococcus furiosus / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1006/abbi.1999.1121 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10087174 ● Summary page in Scopus @ Elsevier: 2-s2.0-0033117895 (DOI: 10.1006/abbi.1999.1121, PubMed: 10087174, Elsevier: Scopus)
65.	Chizu Kujo, Haruhiko Sakuraba, Naoki Nunoura and Toshihisa Ohshima : The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol.1434, No.2, 365-371, 1999. (Summary) The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea. (Keyword) Amino Acid Sequence / Base Sequence / Cloning, Molecular / Electrophoresis, Polyacrylamide Gel / Gene Expression / Genes, Archaeal / Glutamate Dehydrogenase / Molecular Sequence Data / Sequence Alignment / Thermoproteaceae (Link to Publication Site) ● Publication site (DOI): 10.1016/S0167-4838(99)00192-2 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10525154 ● Search Scopus @ Elsevier (PMID): 10525154 ● Search Scopus @ Elsevier (DOI): 10.1016/S0167-4838(99)00192-2 (DOI: 10.1016/S0167-4838(99)00192-2, PubMed: 10525154)
66.	Shin-ichi Sakasegawa, Issei Yoshioka, Shinzi Koga, Mamoru Takahashi, Kunio Matsumoto, Hideo Misaki and Toshihisa Ohshima : A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization, Gene Cloning and Primary Structure, Bioscience, Biotechnology, and Biochemistry, Vol.62, No.12, 2388-2395, 1998.
67.	Chizu Kujo and Toshihisa Ohshima : Enzymological Characteristics of the Hyperthermostable NAD-Dependent Glutamate Dehydrogenase from the Archaeon Pyrobaculum islandicum and Effects of Denaturants and Organic Solvents, Applied and Environmental Microbiology, Vol.64, No.6, 2152-2157, 1998.
68.	Andrey Galkin, Ljudmila Kulakova, Toshihisa Ohshima, Nobuyoshi Esaki and Kenji Soda : Construction of a new leucine dehydrogenase with preferred specificity for NADP⁺ by site-directed mutagenesis of the strictly NAD⁺-specific enzyme, Protein Engineering, Vol.10, No.6, 687-690, 1997. (Summary) On the basis of sequence comparison between NAD+-dependent leucine dehydrogenase (LeuDH) from Thermoactinomyces intermedius and NADP+-dependent dehydrogenases, a set of amino acid residues that are supposed to determine the coenzyme specificity of LeuDH were assigned. Systematic replacement of these amino acids by others was done with the aim to switch its natural coenzyme specificity to a new one preferring NADP+. Single D203A, double D203A-I204R and triple D203A-I204R-D210R mutation enzymes were constructed. The wild-type LeuDH is inactive with NADP+. However, D203A single mutant exhibited dual specificity for NAD+ and NADP+ with essentially identical k(cat)/Km values for both coenzymes, but the values were three orders of magnitude lower than that of the wild-type enzyme. Introduction of positive charge at 204 together with the removal of the negative charge at 203 in the double mutant D203A-I204R provided the enzyme with significantly high affinity for NADP+. The best k(cat)/Km value for NADP+ was shown for the triple mutant D203A-I204R-D210R: more than 2% of the k(cat)/Km value of the wild-type enzyme. Thus, we succeeded in constructing a mutant LeuDH with a new coenzyme specificity preferring NADP+ which is highly active (specific activity, 19 micromol/mg/min). (Keyword) Amino Acid Oxidoreductases / Genetic Vectors / Kinetics / Leucine Dehydrogenase / Micromonosporaceae / Mutagenesis, Site-Directed / NAD / NADP / protein engineering / structureactivity relationship (Link to Publication Site) ● Publication site (DOI): 10.1093/protein/10.6.687 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 9278282 ● Search Scopus @ Elsevier (PMID): 9278282 ● Search Scopus @ Elsevier (DOI): 10.1093/protein/10.6.687 (DOI: 10.1093/protein/10.6.687, PubMed: 9278282)
69.	Beong-Sam Jeon, Hayao Taguchi, Hiroshi Sakai, Toshihisa Ohshima, Takayoshi Wakagi and Hiroshi Matsuzawa : 4-α-Glucanotransferase from the hyperthermophilic archaeon Thermococcus litoralis., --- Enzyme purification and characterization, and gene cloning, sequencing and expression in Escherichia coli ---, European Journal of Biochemistry, Vol.248, No.4, 171-178, 1997. (Summary) 4-Alpha-Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optimal temperature for its activity was 90 degrees C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive saccharides were produced. On incubation of amylose with the T. litoralis enzyme, glucoamylase-resistant but alpha-amylase-sensitive molecules were produced, but the amount of reducing sugar showed only slight increases. These results indicate that the T. litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear alpha-1,4-glucan, but also intramolecular transglycosylation to produce cyclic alpha-1,4-glucan (cycloamylose), similarly to potato 4-alpha-glucanotransferase (called disproportionating enzyme). The gene encoding the T. litoralis 4-alpha-glucanotransferase was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77,883 Da. The amino acid sequence of the T. litoralis enzyme showed high similarity with those of alpha-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-alpha-glucanotransferases. (Keyword) Amino Acid Sequence / Archaea / Base Sequence / Cloning, Molecular / DNA Primers / DNA, Bacterial / Escherichia coli / Gene Expression / Genes, Bacterial / Glycogen Debranching Enzyme System / Hot Temperature / Kinetics / Molecular Sequence Data / Molecular Weight / Polymerase Chain Reaction / Recombinant Proteins / Sequence Homology, Amino Acid / Substrate Specificity / alpha-Amylases (Link to Publication Site) ● Publication site (DOI): 10.1111/j.1432-1033.1997.00171.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 9310375 ● Summary page in Scopus @ Elsevier: 2-s2.0-0030738673 (DOI: 10.1111/j.1432-1033.1997.00171.x, PubMed: 9310375, Elsevier: Scopus)
70.	Kohji Ishihara, Miho Nishitani, Hitomi Yamaguchi, Nobuyoshi Nakajima, Toshihisa Ohshima and Kaoru Nakamura : Preparation of Optically Active α-Hydroxy Esters: Stereoselective Reduction of α-Keto Esters Using Thermophilic Actinomycetes., Journal of Fermentation and Bioengineering, Vol.84, No.3, 268-270, 1997.
71.	Kohji Ishikawa, Miho Nishitani, Hitomi Yamaguchi, Nobuyoshi Nakajima, Toshihisa Ohshima and Kaoru Nakayama : Stereoselective reduction of α- and β-keto esters with aerobic thermophiles, Bacillus strains., Bioscience, Biotechnology, and Biochemistry, Vol.60, 1896-1898, 1996.
72.	Koji Teshima, Toshihisa Ohshima, Satohiro Tanaka and Toshiyuki Nagai : Biomechanical effects of shock waves on Escherichia coli and λphaseDNA., Shock Waves, Vol.4, 293-297, 1995.
73.	Noguchi Akiko, Aoki Toyoaki and Toshihisa Ohshima : Determination of Phosphate Using Immobilized Enzyme Reactor -- Chemiluminescence Detector by FIA, Journal of Flow Injection Analysis, Vol.12, 209-215, 1995.
74.	Shinji Nagata, Sundararaju Bakthavatsulam, Andrey G Galkin, Hiroshi Asada, Sachiko Sakai, Nobuyoshi Esaki, Kenji Soda, Toshihisa Ohshima, Susumu Nagasaki and Haruo Misono : Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9., Appl. Microbiol. Biotechnol., Vol.44, 432-438, 1995.
75.	松美 Oshima and Toshihisa Ohshima : 食品甘味料としての異性化糖の教材化, Chemistry and Education, Vol.43, 710-713, 1995.
76.	Kunishige Kataoka, Harumi Takada, Katsuyuki Tanizawa, Tohru Yoshimura, Nobuyoshi Esaki, Toshihisa Ohshima and Kenji Soda : Construction and Characterization of Chimeric Enzyme Consisting of an Amino-Terminal Domain of Phenylalanine Dehydrogenase and a Carboxy-Terminal Domain of Leucine Dehydrogenase, The Journal of Biochemistry, Vol.116, 931-936, 1994.
77.	Toshihisa Ohshima, Norikazu Nishida, Sundararaju Bakthavatslam, Kunishige Kataoka, Harumi Takada, Tohru Yoshimura, Nobuyuki Esaki and Kenji Soda : The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius, European Journal of Biochemistry, Vol.222, No.2, 305-312, 1994. (Summary) Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4 degrees C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KCl. It retained about 90% of activity on incubation at 70 degrees C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, L-leucine, NADH, 2-oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 118 mM, respectively, from initial-velocity analyses. The enzyme showed pro-S stereospecificity for hydrogen transfer of NADH in the reductive amination. The enzyme gene was cloned into Escherichia coli and its complete DNA sequence was determined. The leucine dehydrogenase gene (leudh) consists of a 1098-bp open reading frame and encodes 366 amino acid residues corresponding to a subunit (M(r) 40586) of the octameric enzyme. The amino acid sequence of the enzyme showed 80.7% similarity with that of the Bacillus stearothermophilus enzyme. The enzyme was overproduced in E. coli JM 109 having a recombinant plasmid, pULDH2, which was constructed from pUC18 and the leudh gene. The enzyme was purified from the cell extract to homogeneity in one day, with 78% recovery. (Keyword) Actinomyces / Amino Acid Oxidoreductases / Amino Acid Sequence / Base Sequence / Chromatography, Affinity / Chromatography, Gel / Cloning, Molecular / Electrophoresis, Polyacrylamide Gel / Enzyme Stability / Escherichia coli / Genes, Bacterial / Hot Temperature / Hydrogen-Ion Concentration / Kinetics / Leucine Dehydrogenase / Magnetic Resonance Spectroscopy / Molecular Sequence Data / Molecular Weight / NAD / Recombinant Proteins / Stereoisomerism / Substrate Specificity / Thermodynamics / Time Factors (Link to Publication Site) ● Publication site (DOI): 10.1111/j.1432-1033.1994.tb18869.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 8020469 ● Summary page in Scopus @ Elsevier: 2-s2.0-0028360284 (DOI: 10.1111/j.1432-1033.1994.tb18869.x, PubMed: 8020469, Elsevier: Scopus)
78.	Toshihisa Ohshima and Norikazu Nishida : Purification and Characterizaion of Extremely Thermo-Stable Glutamate Dehydrogenase from a Hyperthermophilic Archaeon, Thermococcus Litoralis, Biocatalysis, Vol.11, 117-129, 1994.
79.	大島松美, 田中悟広 and Toshihisa Ohshima : ポリアクリルアミド電気泳動法を利用する酵素の特異性を調べる実験の教材化, Chemistry and Education, Vol.42, 137-141, 1994.
80.	Toshihisa Ohshima, 西田憲一, 大島松美 and 山崎隆 : 色素アフィニティクロマトグラフィーの生化学実習のための教材化, Chemistry and Education, Vol.41, 251-254, 1993.
81.	Kunishige Kataoka, Harumi Takada, Tohru Yoshimura, Setsuo Furuyoshi, Nobuyoshi Esaki, Toshihisa Ohshima and Kenji Soda : Site-Directed Mutagenesis of a Hexapeptide Segment Involved in Substrate Recognition of Phenylalanine Dehydrogenase from Thermoactinomyces intermedius, The Journal of Biochemistry, Vol.114, No.1, 69-75, 1993.
82.	Toshihisa Ohshima and Norikazu Nishida : Purification and Properties Extremely Thermostable Glutamate Dehydrogenase from Two Hyperthermophilic Archaebacteria, Pyrococcus woesei and Pyrococcus furiosus, Bioscience, Biotechnology, and Biochemistry, Vol.57, 945-951, 1993.
83.	Toshihisa Ohshima and Satohiro Tanaka : Dye-linked L-malate dehydrogenase from thermophilic Bacillus species DSM 465 Purification and characterization, European Journal of Biochemistry, Vol.214, No.1, 37-42, 1993. (Summary) The distribution of dye-linked L-malate dehydrogenase (L-malate: acceptor oxidoreductase, EC 1.1.99.16) was investigated in many thermophilic bacteria. The enzyme occurred widely in thermophilic spore-forming bacteria like bacilli and thermoactinomycetes. The enzyme was purified to homogeneity from a thermophile, Bacillus sp. DSM 465, with a 2.7% overall recovery by DEAE-Toyopearl column chromatography, Sephacryl S-400 column chromatography and preparative slab PAGE. The enzyme had a molecular mass of about 660 kDa and consisted of about ten subunits all with a molecular mass of 66 kDa. The enzyme retained its full activity upon heating at 55 degrees C for at least 60 min and with incubation at pH 5.0-10.0, 55 degrees C, for 10 min. The enzyme exclusively catalyzed L-malate dehydrogenation in the presence of an electron acceptor such as 2,6-dichloroindophenol. The Michaelis constants for L-malate and 2,6-dichloroindophenol were determined to be 1.67 mM and 0.050 mM, respectively. FAD was identified as a prosthetic group of the enzyme by HPLC. (Keyword) Bacillus / Chromatography, Gel / Chromatography, High Pressure Liquid / Coloring Agents / Electrophoresis, Polyacrylamide Gel / Enzyme Stability / Hydrogen Peroxide / Hydrogen-Ion Concentration / Malate Dehydrogenase / Molecular Weight / Oxaloacetates / Oxygen Consumption / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1111/j.1432-1033.1993.tb17893.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 8508804 ● Summary page in Scopus @ Elsevier: 2-s2.0-0027324398 (DOI: 10.1111/j.1432-1033.1993.tb17893.x, PubMed: 8508804, Elsevier: Scopus)
84.	Toshihisa Ohshima and Kenji Soda : Valine dehydrogenase from a non-spore-forming bacterium, Alcaligenes faecalis: purification and characterization, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol.1162, No.1-2, 221-226, 1993. (Summary) An NAD-dependent valine dehydrogenase (L-valine:NAD oxidoreductase, deaminating, EC 1.4.1.-), was found in a non-spore-forming bacterium, Alcaligenes faecalis, and purified about 80-fold to be characterized. The molecular mass of the enzyme was estimated to be about 72 kDa and the enzyme consists of two identical subunits with a molecular mass of 40 kDa and is thermolabile. It loses its activity fully on incubation at 50 degrees C for 5 min. The enzyme catalyzes the reversible deamination of L-valine, which is the preferred substrate, and other branched-chain and straight-chain L-amino acids in the presence of NAD. The pH optima are about 10.8 and 8.8 for the oxidative deamination and reductive amination, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate in the reductive amination. The amino-acid composition markedly differs from those of Bacillus sphaericus and Clostridium thermoaceticum leucine dehydrogenases. The enzyme did not react with the antibody of C. thermoaceticum leucine dehydrogenase. Therefore, the enzyme is clearly different from leucine dehydrogenases from spore-forming bacteria, which act on valine, and the first valine dehydrogenase to be characterized in detail. (Keyword) Alcaligenes / Amino Acids / Enzyme Stability / Hydrogen-Ion Concentration / Leucine / Molecular Weight / NAD / Oxidoreductases / Substrate Specificity / Valine (Link to Publication Site) ● Publication site (DOI): 10.1016/0167-4838(93)90151-G (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 8448188 ● Search Scopus @ Elsevier (PMID): 8448188 ● Search Scopus @ Elsevier (DOI): 10.1016/0167-4838(93)90151-G (DOI: 10.1016/0167-4838(93)90151-G, PubMed: 8448188)
85.	Toshihisa Ohshima and Masahiko Ishida : A Large-Scale Preparative Electrophoretic Method for the Purification of Pyridine Nucleotide-Linked Dehydrogenases, Protein Expression and Purification, Vol.3, No.2, 121-125, 1992. (Summary) A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography. (Keyword) Alanine Dehydrogenase / Amino Acid Oxidoreductases / Bacterial Proteins / Electrophoresis, Polyacrylamide Gel / Leucine Dehydrogenase / NAD / NADP / Oxidoreductases / Sugar Alcohol Dehydrogenases (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 1422215 ● Search Scopus @ Elsevier (PMID): 1422215 (PubMed: 1422215)
86.	Toshihisa Ohshima, Tanaka Satohiro and Teshima Koji : Effect of Shock Waves on Microorganisms: Evaluation Methods of the Effects; Shock Waves, Proceedings of 18th International Synposium on Shock Waves, Sendai, Springer- Verlag, 1215-1219, 1992.
87.	Toshihisa Ohshima, Harumi Takada, Toru Yoshimura, Nobuyoshi Esaki and Kenji Soda : Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes., Journal of Bacteriology, Vol.173, No.13, 3943-3948, 1991. (Summary) Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate. (Keyword) Actinomycetales / Amino Acid Oxidoreductases / Energy Transfer / Enzyme Stability / Hot Temperature / Hydrogen / Hydrogen-Ion Concentration / Kinetics / Molecular Weight / Phenylalanine / Substrate Specificity (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 2061279 ● Search Scopus @ Elsevier (PMID): 2061279 (PubMed: 2061279)
88.	Harumi Takada, Toru Yoshioka, Toshihisa Ohshima, Nobuhiko Esaki and Kenji Soda : Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius, --- cloning, expression, and sequencing of its gene ---, The Journal of Biochemistry, Vol.109, 371-376, 1991.
89.	Toshihisa Ohshima, Masaru Sakane, Takashi Yamazaki and Kenji Soda : Thermostable Alanine Dehydrogenase from Thermophilic Bacillus sphaericus DSM 462, European Journal of Biochemistry, Vol.191, No.3, 715-720, 1990. (Summary) Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM. (Keyword) Alanine Dehydrogenase / Amino Acid Oxidoreductases / Bacillus / Chromatography / Electrophoresis, Polyacrylamide Gel / Enzyme Stability / Fractional Precipitation / Hot Temperature / Hydrogen-Ion Concentration / Kinetics / Molecular Weight / NAD / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1111/j.1432-1033.1990.tb19180.x (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 2390993 ● Summary page in Scopus @ Elsevier: 2-s2.0-0024987320 (DOI: 10.1111/j.1432-1033.1990.tb19180.x, PubMed: 2390993, Elsevier: Scopus)
90.	Toshihisa Ohshima, Christian Wandrey and Dietrich Conrad : Continous Production of 3-Fluoro-L-alanine with Alanine Dehydrogenase, Biotechnology and Bioengineering, Vol.34, 394-397, 1989.
91.	Nobuyoshi Esaki, Hiroko Shimoi, Nobuyoshi Nakajima, Toshihisa Ohshima, Hidehiko Tanaka and Kenji Soda : Enzymatic in Situ Determination of Stereospecificity of NAD-dependent Dehydrogenases, The Journal of Biological Chemistry, Vol.264, No.17, 9750-9752, 1989.
92.	Shinji Nagata, Katsuyuki Tanizawa, Nobuhiko Esaki, Yonekazu Sakamoto, Toshihisa Ohshima, Hidehiko Tanaka and Kenji Soda : Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus, and structural comparison with other NAD(P)+ dependent dehydrogenase., Biochemistry, Vol.27, 9056-9062, 1988.
93.	Toshihisa Ohshima, Hiroko Sugimoto and Kenji Soda : Selective enzymatic determination of L-phenylalanine and 3-phenylpyruvate with phenylalanine dehydrogenase., Analytical Letters, Vol.21, 2205-2215, 1988.
94.	Toshihisa Ohshima : NAD要求性アミノ酸脱水素酵素の酵素化学的研究とその応用, Vitamins, Vol.61, 535-548, 1987.
95.	Tomoya Takahashi, Takahito Kondo, Hideki Ohno, Sadamasa Minato, Toshihisa Ohshima, Shuzei Mikuni, Kenji Soda and Naoyuki Taniguchi : A spectrophotometric method for the determination of γ-glutamyl cyclotransferase with analine dehydrogenase in the presence of anthglutin., Biochemical Medicine and Metabolic Biology, Vol.38, 311-316, 1987.
96.	Toshihisa Ohshima and Haruhiko Sakuraba : Purification and characterization of malate dehydrogenase from the phototrophic bacterium, Rhodopseudomonas capsulata, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol.869, No.2, 171-177, 1986. (Summary) 光合成細菌Rhodopseudomonas capsulataのエネルギー代謝と炭酸固定に関与するクエン酸サイクルの酵素リンゴ酸脱水素酵素の精製と性質について明らかにした．リンゴ酸脱水素酵素の新規反応メカニズムを提唱した． (Link to Publication Site) ● Publication site (DOI): 10.1016/0167-4838(86)90291-8 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/0167-4838(86)90291-8 (DOI: 10.1016/0167-4838(86)90291-8)
97.	Toshihisa Ohshima, Nagata Shinji and Soda Kenji : Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus, Arch. Microbiol, Vol.141, 407-411, 1985.
98.	Toshihisa Ohshima, Wandrey Christian, Kula Maria-Regina and Soda Kenji : Improvement for L-Leucine Production in a Continuously Operated Enzyme Membrane Reactor, Biotechnol. Bioeng, Vol.27, 1616-1618, 1985.
99.	Toshihisa Ohshima, Wandrey Christian, Sugiura Miyuki and Soda Kenji : SCREENING OF THERMOSTABLE LEUCINE AND ALANINE DEHYDROGENASES IN THERMOPHILIC BACILLUS STRAINS, Biotechnol. Lett, Vol.7, 871-876, 1985.
100.	Toshihisa Ohshima, Ohshima Matsumi and Drews Gerhart : Purification and Partial Characterization of the Soluble NADH Dehydrogenase from the Phototrophic Bacterium, Rhodopseudomonas capsulata. Z. Naturforsch, Vol.39, 68-72, 1984.
101.	Toshihisa Ohshima and Soda Kenji : Modification of Leucine Dehydrogenase by Pyridoxal 5'-Phosphate., Agricultural and Biological Chemistry, Vol.48, 349-354, 1984.
102.	Ohtawara Shigeki, Toshihisa Ohshima, Esaki Nobuyoshi and Soda Kenji : Purification and Properties of α;-Hydroxyglutarate Dehydrogenase of Peptococcus aerogenes. Agric., Biol. Chem, Vol.48, 1713-1719, 1984.
103.	Takamiya Shizuo, Toshihisa Ohshima, Tanizawa Katsuyuki and Soda Kenji : A Spectrophotometric Method for the Determination of Aminopeptidase Activity with Leucine Dehydrogenase, Analytical Biochemistry: Methods in the Biological Sciences, Vol.130, No.1, 266-270, 1983. (Keyword) leucine dehydrogenase / aminopeptidase / leucine aminopeptidase / amino acid arylamidase / leucine amide / leucine peptide (Link to Publication Site) ● Publication site (DOI): 10.1016/0003-2697(83)90678-4 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/0003-2697(83)90678-4 (DOI: 10.1016/0003-2697(83)90678-4)
104.	Toshihisa Ohshima, Itoh Satoshi and Soda Kenji : A Rapid Method for Purification of Leucine Dehydrogenase by Affinity Chromatography., Bulletin of the Institute for Chemical Research Kyoto University, Vol.61, 109-112, 1983.
105.	Takamiya Shizuo, Toshihisa Ohshima, Tanizawa Katsuyuki and Soda Kenji : Spectrophotometric End Point Method for Assaying Serum Aminopeptidase, Agricultural and Biological Chemistry, Vol.47, 893-895, 1983.
106.	Hiragi Yuzuru, Soda Kenji and Toshihisa Ohshima : Small-Angle X-ray Scattering Studies of L-Leucine Dehydrogenase., Makromol. Chem, Vol.183, 745-751, 1982.
107.	Toshihisa Ohshima and Drews Gerhart : Isolation and Partial Characterization of the Membrane-bound NADH Dehydrogenase from Phototrophic Bacterium Rhodopseudomonas capsulata, Z. Naturforsch, Vol.36, 400-406, 1981.
108.	Toshihisa Ohshima and Soda Kenji : Purification and Properties of Alanine Dehydrogenase from Bacillus sphaericus., European Journal of Biochemistry, Vol.100, No.1, 29-39, 1979.
109.	Toshihisa Ohshima, Haruo Misono and Kenji Soda : Properties of Crystalline Leucine Dehydrogenase from Bacillus sphaericus., The Journal of Biological Chemistry, Vol.253, No.16, 5719-5725, 1978.
110.	Toshihisa Ohshima, Yamamoto Tatsuo, Misono Haruo and Soda Kenji : Leucine Dehydrogenase of Bacillus sphaericus: Sulfhydryl Groups and Catalytic Sites., Agricultural and Biological Chemistry, Vol.42, 1739-1743, 1978.
111.	Toshihisa Ohshima, Misono Haruo and Soda Kenji : Determination of Branched- chain L-Amino Acids and Their Keto Analogs with Leucine Dehydrogenase., Agricultural and Biological Chemistry, Vol.42, 1919-1922, 1978.
112.	Soda Kenji, Misono Haruo, Toshihisa Ohshima, Yamamoto Tatsuo and Oki Toshikazu : A BACTERIAL ANTITUMOR ENZYME, LEUCINE DEHYDROGENASE FROM BACILLUS SPHAERICUS, PROCEEDINGS OF THE FIRST INTERNATIONAL CONGRESS OF IAMS, Vol.5, 266-270, 1975.

Academic Paper (Unrefereed Paper):

1.	Shuichiro Goda, Tomoki Yabutani, Haruhiko Sakuraba, Junko Motonaka and Toshihisa Ohshima : Development of Biosensors Using Hyperthermophilic Enzymes as an Element:Development of Biosensors Using Hyperthermophilic Enzymes as an Element:Functional Analysis of Polyamine Metabolizing Enzymes and Development for EnzymeElectrode Sensor Using D-Proline Dehydrogenases, Bulletin of Faculty of Engineering, The University of Tokushima, Vol.50, 2005. (Summary) An amperometric enzyme sensor give us higher sensitivity and specificity for the substratedetermination. In spite of advantages of enzyme sensor, many enzymes so far found have been toolabile to use as biosensor elements in artificial circumstances for a longer period.Hyperthermophiles, which can grow above 90℃, have been known to produce much more stableenzymes under various artificial conditions. In this work, we carried out screening, biochemicalcharacterization and improvement of production for hyperthermostable enzymes which are more usefulas the elements in the biosensors. We focused on the polyamines as one of the substrates ofbiosensors. Polyamines have been known to play many important roles in cell proliferation,differentiation and transformation. The concentration of the polyamines together with their acetylconjugates remarkably increases in the biological fluids and the affected tissues of cancer patients.Therefore, their polyamines are listed as tumor markers. Gas and ion chromatographies have beenso far used for polyamine determination, but have some problems from the aspects of high sensitivityand easy operation. Thus, we here developed biosensors using hyperthermostable enzymes forpolyamine determination. Such enzyme sensors are more useful for the simple and rapiddetermination of polyamines and application for clinical analysis and food analysis In addition, wetried the construction of biosensor using the hyperthermophilic enzyme, D-Proline dehydrogenase.As the results, we found the thermostable agmatinase and spermidine dehydrogenase inhyperthermophiles, Pyrococcus horikoshii and Sulfolobus tokodaii, respectively. We succeeded theconstruction of novel amperometric sensor for D-proline determination using D-Proline dehydrogenasederived from Pyrobaculum islandicum. (Keyword) enzyme sensor, hyperthermophile, dehydrogenase, polyamine (Tokushima University Institutional Repository) ● Metadata: 45160 (Link to Search Site for Scientific Articles) ● CiNii @ National Institute of Informatics (CRID): 1050001337463675520 (Tokushima University Institutional Repository: 45160, CiNii: 1050001337463675520)

Review, Commentary:

1.	Haruhiko Sakuraba, Shuichiro Goda, Ryushi Kawakami, Toshihisa Ohshima, Hideaki Tsuge and 勝沼信彦 : Molecular Strategy for Thermostability of Hyperthermophilic Protein, 化学と生物, Vol.44, No.5, 305-312, May 2006. (Link to Publication Site) ● Publication site (DOI): 10.1271/kagakutoseibutsu1962.44.305 (Link to Search Site for Scientific Articles) ● CiNii @ National Institute of Informatics (CRID): 1571417125313316352 ● Search Scopus @ Elsevier (DOI): 10.1271/kagakutoseibutsu1962.44.305 (DOI: 10.1271/kagakutoseibutsu1962.44.305, CiNii: 1571417125313316352)
2.	Hideaki Tsuge, Ryushi Kawakami, Haruhiko Sakuraba, 安藝謙嗣, 勝沼信彦 and Toshihisa Ohshima : Novel diflavin-containing dehydrogenase family : FAD, FMN and ATP-containing L-proline dehydrogenase, Seikagaku, Vol.78, No.1, 41-45, Jan. 2006. (Keyword) Adenosine Triphosphate / Crystallization / Flavin Mononucleotide / Flavin-Adenine Dinucleotide / Proline Oxidase / Protein Conformation / Pyrococcus horikoshii / Sarcosine Oxidase (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16491912 ● CiNii @ National Institute of Informatics (CRID): 1520853832706586496 ● Search Scopus @ Elsevier (PMID): 16491912 (PubMed: 16491912, CiNii: 1520853832706586496)
3.	Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアの新規フラビン含有アミノ酸脱水素酵素の機能開発, 酵素工学ニュース, Vol.54, 31-36, Oct. 2005.
4.	Haruhiko Sakuraba, Toshihisa Ohshima, Hideaki Tsuge and 勝沼信彦 : 超好熱菌由来アルドラーゼのユニークな4次構造と高度耐熱性, Seibutsu Butsuri, Vol.45, No.2, 97-100, Mar. 2005.
5.	Toshihisa Ohshima and 高木博史 : 高機能エンザイム創製へのニューアプローチ, 日本農芸化学会誌, Vol.78, No.8, 744, Aug. 2004.
6.	Toshihisa Ohshima, Haruhiko Sakuraba and Hideaki Tsuge : 超好熱菌の酵素の構造解析に基づく新規耐熱性酵素の創製, 日本農芸化学会誌, Vol.78, No.8, 761-763, Aug. 2004. (Summary) 超好熱古細菌のNAD依存性グルタミン酸脱水素酵素のX線結晶構造に基づき，基質結合部位のアミノ酸残基を変異させ，新規高度耐熱性ノルバリン脱水素酵素の創製に成功した結果をまとめている．
7.	Toshihisa Ohshima : 巻頭言:大学法人化と進化, 極限環境微生物学会誌, Vol.3, No.1, 2-3, Jul. 2004.
8.	Haruhiko Sakuraba, Shuichiro Goda and Toshihisa Ohshima : Unique sugar metabolism and novel enzymes of hyperthermophilic archaea, Chemical Record, Vol.3, No.5, 281-287, May 2004. (Summary) Hyperthermophiles are a group of microorganisms that have their optimum growth temperature above 80 degrees C. More than 60 species of the hyperthermophiles have been isolated from marine and continental volcanic environments. Most hyperthermophiles belong to Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed the presence of unusual sugar metabolic processes in hyperthermophilic archaea, for example, a modified Embden-Meyerhof pathway, that has so far not been observed in bacteria and eucarya. Several novel enzymes, such as ADP-dependent glucokinase, ADP-dependent phosphofructokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate : ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase, have been found to be involved in a modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a unique mode of ATP regeneration has been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution. (Keyword) Amino Acid Sequence / Archaea / Carbohydrate Metabolism / Glucokinase / Models, Molecular / Molecular Sequence Data / Phosphotransferases (Alcohol Group Acceptor) / Phylogeny / Sequence Alignment / Sequence Homology, Amino Acid (Link to Publication Site) ● Publication site (DOI): 10.1002/tcr.10066 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 14762828 ● Search Scopus @ Elsevier (PMID): 14762828 ● Search Scopus @ Elsevier (DOI): 10.1002/tcr.10066 (DOI: 10.1002/tcr.10066, PubMed: 14762828)
9.	Haruhiko Sakuraba and Toshihisa Ohshima : Modified Embden-Meyerhof pathway of hyperthermophilic Archaea, Tanpakushitsu Kakusan Koso, Vol.48, No.9, 1256-1262, Jul. 2003. (Summary) 超好熱古細菌の解糖系に存在するADP依存性グルコキナーゼの遺伝子クローニング，AMPからATPを合成する新規キナーゼの発見，その酵素化学的諸性質および遺伝子の転写誘導についての知見をまとめ，これまで不明であった超好熱古細菌のエネルギー代謝におけるこれらの酵素の役割を解説した． (Keyword) Adenosine Triphosphate / Amino Acid Sequence / Energy Metabolism / Glucokinase / Glycolysis / Molecular Sequence Data / Phosphotransferases (Alcohol Group Acceptor) / Pyrococcus furiosus / Sequence Homology, Amino Acid (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12868410 ● Search Scopus @ Elsevier (PMID): 12868410 (PubMed: 12868410)
10.	里村武範 and Toshihisa Ohshima : 色素依存性アミノ酸脱水素酵素の新展開, Seibutsu-kogaku Kaishi, Vol.81, No.3, 121, Mar. 2003. (Link to Search Site for Scientific Articles) ● CiNii @ National Institute of Informatics (CRID): 1543668945055620096 (CiNii: 1543668945055620096)
11.	Haruhiko Sakuraba and Toshihisa Ohshima : 最古の糖代謝系?-超好熱菌が持つユニークなエネルギー代謝-, Seibutsu-kogaku Kaishi, Vol.81, No.1, 20, Jan. 2003. (Summary) 超好熱古細菌の解糖系に存在する2種のADP依存性グルコキナーゼの遺伝子クローニングおよびAMPからATPを合成する新規キナーゼの発見について解説した． (Link to Search Site for Scientific Articles) ● CiNii @ National Institute of Informatics (CRID): 1542824520125485056 (CiNii: 1542824520125485056)
12.	Haruhiko Sakuraba and Toshihisa Ohshima : Novel energy metabolism in anaerobic hyperthermophilic archaea: the modified Embden-Meyerhof pathway, Journal of Bioscience and Bioengineering, Vol.93, No.5, 441-448, Mar. 2002. (Summary) Hyperthermophiles, a group of microorganisms whose optimum growth temperatures are above 80 degrees C, have been isolated mainly from marine and continental volcanic environments. They are viewed as potential sources of extraordinarily stable biomolecules with applications in novel industrial processes. Most hyperthermophiles belong to the domain Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed unusual energy metabolic processes in hyperthermophilic archaea, e.g. a modified Embden-Meyerhof pathway, that have not been observed so far in organisms belonging to the Bacteria and Eucarya domains. Several novel enzymes--ADP-dependent glucokinase, ADP-dependent phosphofruktokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate: ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase--have been found to be involved in the modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a novel regulation site for energy metabolism and a unique mode of ATP regeneration have been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution. This review focuses mainly on the unique energy metabolism and related enzymes of P. furiosus that have recently been described. (Link to Publication Site) ● Publication site (DOI): 10.1016/S1389-1723(02)80090-1 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 16233230 ● Summary page in Scopus @ Elsevier: 2-s2.0-0036082038 (DOI: 10.1016/S1389-1723(02)80090-1, PubMed: 16233230, Elsevier: Scopus)
13.	Haruhiko Sakuraba and Toshihisa Ohshima : ゲノム情報からみえた超好熱菌のNAD生合成系, 化学と生物, Vol.39, No.12, 774-776, Dec. 2001. (Summary) ゲノム情報に基づき，超好熱菌に初めて見出したL-アスパラギン酸オキシダーゼが嫌気条件下でも機能する色素依存性L-アスパラギン酸脱水素酵素反応も触媒することを見出し，NAD生合成系の初発酵素として機能していることを提唱した．
14.	Haruhiko Sakuraba and Toshihisa Ohshima : Hyperthermophiles-their unique energy metabolism and enzymes, バイオサイエンスとインダストリー, Vol.59, No.7, 11-16, Jan. 2001. (Summary) 超好熱古細菌Pyrococcus furiosusに初めて見い出した新規キナーゼ(ホスホエノールピルビン酸とAMPからATPを合成する反応を触媒する)の酵素化学的諸性質について論述し，これまで不明であった超好熱古細菌のエネルギー代謝における本酵素の新しい役割を提唱した．
15.	関口順一, 青山紘一, Toshihisa Ohshima, 大島淳, 河原秀久, 清水和幸, 西村基弘, 播磨武 and 室岡義勝 : 世界をリードする生物工学教育とは, Seibutsu-kogaku Kaishi, Vol.79, 470-489, 2001.
16.	Toshihisa Ohshima : 嫌気性超好熱古細菌Pyrococcus furiosusに見られる珍しい解糖系, Seibutsu-kogaku Kaishi, Vol.78, No.9, 403, Jan. 2000.
17.	公庄千寿, Haruhiko Sakuraba and Toshihisa Ohshima : 嫌気性超好熱菌の簡便な培養法, Seikagaku, Vol.72, No.3, 203-206, Jan. 2000. (Summary) 100℃付近の高温条件を生育に必要とする超好熱菌について，誰にでもできる簡便な培養法および培養装置を考案し，培養装置の詳細および各種超好熱菌の培養条件を紹介した．
18.	Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱古細菌 Pyrococcus furiosus に存在する特異的なATP再生酵素, Vitamins, Vol.73, 206-208, Jan. 1999. (Summary) AMPからATPを合成する新規キナーゼの発見，その酵素化学的諸性質および遺伝子の転写誘導についての知見をまとめ，超好熱古細菌のエネルギー代謝における本酵素の役割を解説した．
19.	Toshihisa Ohshima : 超好熱菌の糖代謝，窒素代謝経路の特性, 生物工学, Vol.76, No.6, 263-266, Jan. 1998.
20.	Toshihisa Ohshima : 超好熱菌とその酵素の特性, STEPテクノ情報, Vol.3, No.1, 25-31, Mar. 1997.
21.	Toshihisa Ohshima : 超好熱古細菌Pyrococcus furiosusの新規解糖系, Vitamins, Vol.70, 290-292, 1996.
22.	Toshihisa Ohshima and 松美 Oshima : ゆであがらない酵素, Vitamins, Vol.68, 692-694, 1994.
23.	Toshihisa Ohshima and 福田知彦 : ピリドキサールリン酸によるアミノ酸脱水素酵素の活性部位におけるリシン残基の機能解析, Vitamins, Vol.67, 275-277, 1993.
24.	Toshihisa Ohshima and 福田知彦 : 酵素·タンパク質の分取用スラブゲル電気泳動法, 植物細胞工学, Vol.5, 124-129, 1993.
25.	Toshihisa Ohshima : NAD(P)要求性脱水素酵素の補酵素特異性の変換, Vitamins, Vol.66, 609-611, Jan. 1992.
26.	Toshihisa Ohshima : アミノ酸脱水素酵素の機能開発, 化学と生物, Vol.30, No.10, 656-665, Jan. 1992.
27.	Toshihisa Ohshima : 色素アフィニティクロマトグラフィーの特徴と利用法, Journal of Medical Technology, Vol.35, 317-320, Jan. 1991.
28.	Toshihisa Ohshima and 山崎隆 : 色素リガンドアフィニティークロマトグラフィー ‐吸着体の簡単な調製法と利用, Seikagaku, Vol.62, 283-287, Jan. 1990.
29.	Toshihisa Ohshima and Soda Kenji : Thermostable amino acid dehydrogenases: applications and gene cloning, Trends in Biotechnology, Vol.7, 210-214, Jan. 1989.
30.	Toshihisa Ohshima and Soda Kenji : Biotechnological aspects of amino acid dehydrogenases, International Industrial Biotechnology, Vol.9, 5-11, Jan. 1989.
31.	Toshihisa Ohshima and 坂本弘樹 : 簡単にできる調製用アクリルアミドスラブゲル電気泳動法-脱水素酵素の精製への適用, Seikagaku, Vol.60, 374-377, Jan. 1988.
32.	Toshihisa Ohshima : Bis-NADを用いる脱水素酵素のAffinity precipitation, Vitamins, Vol.60, 423-424, Jan. 1986.
33.	Toshihisa Ohshima and 左右田健次 : 酵素の新しい精製法と利用, ケミカル·エンジニアリング, Vol.31, 395-400, Jan. 1986.
34.	Toshihisa Ohshima and 左右田健次 : ロイシン脱水素酵素, 発酵と工業, Vol.43, 919-926, Jan. 1985.
35.	左右田健次, Toshihisa Ohshima and 江崎信芳 : ライフサイエンスのための安定同位体利用技術(XIII)同位体効果利用による酵素反応研究法, Radioisotopes, Vol.32, 42-52, Jan. 1983.
36.	Toshihisa Ohshima : Studies on Bacterial Leucine Dehydrogenase, 京都大学農学博士論文, 1-72, Jan. 1978.

Proceeding of International Conference:

1.	Yuji Tani, Kenichi Nishi, Chikahiro Wada, Yoshio Shoda, Toshio Watanabe, Haruhiko Sakuraba, Toshihisa Ohshima, Yukako Hayashi, Tomoki Yabutani and Junko Motonaka : Characterization and application of thermostable D-proline dehydrogenase and carbon nanotube gel immobilized electrode, AMDP 2008 (International Conference on Advanced Materials, Development and Performance 2008), 202, Beijing, Oct. 2008.
2.	Yohei Yamada, Yumiko Takeuchi, Yuji Tani, Haruhiko Sakuraba, Toshihisa Ohshima, Shinichi Sakasegawa, Yukako Hayashi, Tomoki Yabutani and Junko Motonaka : Development of an amperometric enzyme sensor based on immobilization of thermostable bilirubin oxidase, AMDP 2008 (International Conference on Advanced Materials, Development and Performance 2008), 144-145, Beijing, Oct. 2008.
3.	Tani Yuji, Tomoki Yabutani, Mishima Yuji, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : Electrochemical characterization of a thermostable D-proline dehydrogenase immobilized electrode under aqueous and alcohol solvents, the 9th Asian Conference on Analytical Chemistry, Jeju(Korea), Nov. 2007.
4.	Natsuko Kusunoki, Kozue Edo, Kaori Ohnishi, Keiko Ashida, Asami Nakanishi, Hitomi Sakai, Hideyuki Nakagawa, Kumio Yokoigawa, Haruhiko Sakuraba and Toshihisa Ohshima : Multiple lectins from globiferous pedicellariae of the sea urchin, Toxopneustes pileolus, The Proceeding of 21st Pacific Science Congress:Session:4-10 Health hazard by marine animals in Pacific region, 7p, Okinawa, Jun. 2007. (Keyword) lectin / sea urchin / venom / Toxopneustes pileolus / pedicellariae
5.	Shimizu Y., Haruhiko Sakuraba and Toshihisa Ohshima : Elucidation of the L-Serine Biosynthesis in the Hyperthermophilic Archaeon:Characterization of D-3-Phosphoglycerate Dehydrogenase from Sulfolobus tokodaii, The first joint Symposium on life between Pusan National Univeristy and Kyushu University, Fukuoka, Feb. 2007.
6.	Fukuda J., Yoneda K., Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : Crystallization and Chracterization of L-lysine Dehydrogenase (LysDH) from Hyperthermophilic Archaeon yrococcus horikoshii, The first joint Symposium on life between Pusan National Univeristy and Kyushu University, Fukuoka, Feb. 2007.
7.	Yoneda K., Ryushi Kawakami, Tagashira Y., Haruhiko Sakuraba, Shuichiro Goda, Hideaki Tsuge, Katunuma N. and Toshihisa Ohshima : The first crystal structure of an archaeal L-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus, EXTREMOPHILES 2006 CONFERENCE, Brest, Sep. 2006.
8.	Toshihisa Ohshima and Haruhiko Sakuraba : Crystal structure of D-2-deoxyribose-5-phosphate aldolase from a hyperthermophilic archaeon Aeropyrum pernix, Pacifichem 2005, Honolulu, Hawaii, USA, Dec. 2005.
9.	Haruhiko Sakuraba, Tsuge Hideaki, Yoneda Kazunari, Katunuma Nobuhiko and Toshihisa Ohshima : The first crystal structure of the NAD biosynthetic enzyme quinolinate synthase, International Interdisciplinary Conference of Vitamins, Coenzyme and Biofactors 2005, Awaji Island, Japan, Nov. 2005.
10.	Yoneda Kazunari, Haruhiko Sakuraba, Tsuge Hideaki, Katunuma Nobuhiko, Kuramitsu Seiki, Kawabata Takeshi and Toshihisa Ohshima : The crystal structure of ST1625p from hyperthermophilic archaeon Sulfolobus tokodaii: a novel fold of helical repeat protein, INTERNATIONAL SYMPOSIUM ON EXTREMOPHILES AND THEIR APPLICATIONS, Tokyo, Nov. 2005.
11.	Toshihisa Ohshima : Structure and function of hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolases, The 13th German-Japanese Workshop on Enzyme Technology, Uberlingen, Germany, Sep. 2005.
12.	Hideaki Tsuge, Ryushi Kawakami, Haruhiko Sakuraba, Kenji Aki, Nobuhiko Katunuma, Hideo Ago, Masashi Miyano and Toshihisa Ohshima : The Crystal Structure of L-proline Dehydrogenase in a Hyperthermophilic Archaeon, XX Congress of the International Union of Crystallography, Vol.27, No.50, C198, Florence, Italy, Aug. 2005. (Keyword) dehydrogenase / flavins / complex structure
13.	Yuji Tani, Tomoki Yabutani, Yuji Mishima, Shuichiro Goda, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : Preparation and electrochemical characteristics of thermostable D-Proline dehydrogenase immobilized electrode, Advanced Materials Development & Performance Conference 2005, Auckland, Jul. 2005.
14.	Toshihisa Ohshima, Ryushi Kawakami and Haruhiko Sakuraba : Gene and primary stuructures, and properties of a novel type of dye-linked FAD-containing L-proline dehydrogenase complex from the hyperthremophilic archaeon Thermococcus profundus, The eighth China-Japan-Korea joint symposium on enzyme engineering, 43-44, Hangzhou, China, Oct. 2004. (Summary) 超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，本酵素がユニークなヘテロテトラマー構造をとること，これらをコードする4種の遺伝子がオペロンを形成することを明らかにした．また，この異種サブユニットの共発現系の構築に成功した．
15.	Yoshihisa Suzuki, Gen Sazaki, Yoshiki Matsuura, Hironori Hondoh, Haruhiko Sakuraba, Toshihisa Ohshima, Kazuo Nakajima and Katsuhiro Tamura : A Molecular Level Study on the Growth Kinetics of Glucose Isomerase Crystals, 3rd International Conference on High Pressure Bioscience and Biotechnology, Rio de Janeiro, Sep. 2004. (Summary) グルコースイソメラーゼ(GI)斜方晶系結晶(I222)の(101)面の成長速度R101 を，過飽和度σ ( σ = ln(C/Ce), C: 溶液濃度，Ce: 溶解度) の異なる溶液に対する結晶サイズの時間変化をその場観察することによって求めた．σに対してR101 をplot すると，100 MPa におけるR101 の方が明らかに速く，加圧によって速度論的に成長が促進される事が分かった．それを2 次元核成長のbirth-and-spread model でfitting した結果，加圧に伴う二次元核の平均ステップレッジ自由エネルギーαの減少がこの促進の原因である事がわかった．この結果をさらに詳細に考察するために，また，液中AFM(Nanoscope IIIa)を用いて常圧における(101)面の微細構造の観察を行ったところ，結晶の対称性を反映した三角形の微細構造が見られた．また，結晶中の結合の強さを見積もるために，既出の結晶構造解析データ(PDB ID 1MNZ)を用いた結晶中のマクロボンド解析を行った結果，tetramer 形成の強い結合と，tetramer 間の弱い結合に分類される事が分かった．AFMによって得られたステップ高さは，tetramerの存在を裏付けるものであった．
16.	Toshihisa Ohshima, Mohammad W Bhuiya, Haruhiko Sakuraba, Takahito Imagawa, Nobuhiko Katunuma and Hideaki Tsuge : The first crystal structure of hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum, Extremophiles2004: 5th International Conference on Extremophiles, Maryland, Sep. 2004. (Summary) 超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のNAD依存性グルタミン酸脱水素酵素について，初めてX線結晶構造解析に成功した．これまでグルタミン酸脱水素酵素の高い熱安定性が，イオンペアネットワークの増加に由来するとされてきた定説に対し，サブユニット間の疎水性相互作用の重要性を指摘した．
17.	Shin-ichiro Suye, Yutaka Hirose, Tomokazu Kimura, Haitao Zheng, Teruo Hori, Hideo Katayama, Ryushi Kawakami and Toshihisa Ohshima : Amperometric DNA sensor using gold electrode modified with polymeric mediator and thermostable dehydrogenase by layer-by-layer adsorption, Nano and Giga Challenges in Microelectronics Symposium and Summer School Reserch and Development Opportunities, Cracow, Sep. 2004.
18.	Yoshimi Nishikawa, Kazunari Yoneda, Hideaki Tsuge, Nobuhiko Katunuma, Haruhiko Sakuraba, Yuzuru Hiragi and Toshihisa Ohshima : Structural study on the heat activation process of recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrobaculum islandicum: analysis by small-angle X-ray scattering, SAX in the 21st Century, Mikazuki-cho, Sep. 2004.
19.	Shuichiro Goda, Haruhiko Sakuraba, Yutaka Kawarabayasi and Toshihisa Ohshima : Cloning, Expression, and Characterization of the First Archaeal Agmatinase from Anaerobic Hyperthermophilic Archaeon Pyrococcus horikoshii, International Congress on Biocatalysis 2004 (biocat 2004), Vol.P115, 246, Hamburg, Aug. 2004.
20.	Hideaki Tsuge, Ryushi Kawakami, Haruhiko Sakuraba, Kazunari Yoneda, Hideo Ago, Masashi Miyano, Kenji Aki, Nobuhiko Katunuma and Toshihisa Ohshima : Crystal structure of an unique FAD and FMN containing enzyme; Dye-linked L-proline dehydrogenase from Pyrococcus horikoshii, The 1st Pacific-Rim International Conference on Protein Science, Yokohama, Apr. 2004. (Summary) 超好熱アーキアPyrococcus horikoshiiに見出した新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，これらをコードする2種の遺伝子の共発現系構築に成功した．さらに本酵素のX線結晶構造解析に成功した．
21.	Mohammad W. Bhuiya, Haruhiko Sakuraba, Toshihisa Ohshima, Takahito Imagawa, Nobuhiko Katunuma and Hideaki Tsuge : Structure of hyperthermostable NAD-specific glutamate dehydrogenase of Pyrobaculum islandicum, The 1st Pacific-Rim International Conference on Protein Science, Yokohama, Apr. 2004. (Summary) 超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のNAD依存性グルタミン酸脱水素酵素について，初めてX線結晶構造解析に成功した．本酵素の補酵素特異性と立体構造の相関を明らかにした．
22.	Ryushi Kawakami, Hideaki Tsuge, Shuichiro Goda, Haruhiko Sakuraba, Nobuhiko Katunuma and Toshihisa Ohshima : Novel dye-linked L-proline dehydrogenase from anaerobic hyperthermophilic archaea, Thermococcus profundus and Pyrococcus horikoshii OT-3, The 1st Pacific-Rim International Conference on Protein Science, Yokohama, Apr. 2004. (Summary) 超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，本酵素がユニークなヘテロテトラマー構造をとることを明らかにした．
23.	Toshihisa Ohshima, Haruhiko Sakuraba, Hideaki Tsuge, Shuichiro Goda and Nobuhiko Katunuma : Function and structure of D-2-deoxyribose-5-phosphate aldolase from a marine hyperthermophilic archaeon, Aeropyrum pernix, Marine Biotechnology Conference 2003, Chiba, Sep. 2003.
24.	Shuichiro Goda, Haruhiko Sakuraba, Mohammad W Bhuiya, Ryushi Kawakami, Seiki Kuramitsu, Yuzuru Hiragi and Toshihisa Ohshima : Heat and Urea Induced Activations by Subunit Rearrangement of Hyperthermophilic Glutamate Dehydrogenase Produced in Escherichia Coli, Thermophiles 2003, Vol.P2.17, 143, Exeter, United Kingom, Sep. 2003. (Summary) 大腸菌内で不活性型として生産された超好熱菌由来グルタミン酸脱水素酵素酵素の熱および尿素による成熟化機構について熱力学的解析を行った．
25.	Haruhiko Sakuraba and Toshihisa Ohshima : ADP-dependent glucokinase/phosphofructokinase, a novel sugar kinase from Methanococcus jannashii, The 4th International Congress on Extremophiles, Naples, Itary, Sep. 2002. (Summary) 超好熱アーキアMethanococcus jannashiiに見出したグルコキナーゼおよびホスホフルクトキナーゼの両活性を持つ新規酵素について報告している．この酵素がグルコキナーゼおよびホスホフルクトキナーゼの起源であることを提唱した．本酵素の発見により，メタン菌による糖からのメタン生産が理論上可能であることを示した．
26.	Ryushi Kawakami, Toshihisa Ohshima and Haruhiko Sakuraba : Novel dye-linked L-prorine dehydrogenase from a hyperthemophilic archaeon, Thermococcus profundus, --- Subunit structure, gene structure and catalytic properties ---, 9th International Symposium on the Genetics of Industrial Microorganisms, Gyeongju, Korea, Jul. 2002. (Summary) 超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，本酵素がユニークなヘテロテトラマー構造をとること，これらをコードする4種の遺伝子がオペロンを形成することを明らかにした．
27.	Takenori Satomura, Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : Purification and characterization of dye-linked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum, International Congress on Biocatalysis, Hamburg, Germany, Jun. 2002. (Summary) 超好熱アーキアPyrobaculum islandicumに初めて見出した新規色素依存性D-プロリン脱水素酵素に関して報告している．本酵素は従来報告されているD-アミノ酸脱水素酵素とは大きく構造や性質が異なることを明らかにした．
28.	Toshihisa Ohshima and Haruhiko Sakuraba : ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic Archaeon Methanococcus jannnashii, Natural products from marine microorganisms, Greifswald, Germany, Jun. 2002. (Summary) 超好熱アーキアMethanococcus jannashiiに見出したグルコキナーゼおよびホスホフルクトキナーゼの両活性を持つ新規酵素について報告している．この酵素がグルコキナーゼおよびホスホフルクトキナーゼの起源であることを提唱した．
29.	Fumihiko Satoh, Hideyuki Nakagawa, Kuniko Nagasaka, Toshinori Nagasaka, Haruhiko Sakuraba and Toshihisa Ohshima : Fishing for Bioactive Compounds from Some Marine Vertebrate and Invertebrates, 7th Eurasia Conference on Chemical Sciences, Karachi, Pakistan, Mar. 2002. (Summary) We suggested that somespecies of scorpionfish and the toxopneustid sea urchins may be a resource for biologically active substances. (Keyword) scorpionfish / toxoneustid sea urchins / mitogenic activity / cytotoxicity
30.	Toshihisa Ohshima, Haruhiko Sakuraba, Takenori Satomura and Ryushi Kawakami : Two novel dye-linked proline dehydrogenases from anaerobic hyperthermophilic archaea: Their function, structure and application, Japan-Italy Symposium New Trends in Enzyme Science and Technology, Kyoto, Nov. 2001. (Summary) 超好熱アーキアに見出した2種類の新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，これらをコードする遺伝子がオペロンを形成することを明らかにした．
31.	Toshihisa Ohshima and Haruhiko Sakuraba : Molecular characterization and a role in the de novo biosynthesis of NAD of L-aspartate oxidase from hyperthermophilic archaeon Pyrococcus horikoshii OT-3 on the basis of genome informatics, The 1st International On-Board Seminar for the Development of Environment-friendly Technology, Tokushima, Oct. 2001. (Summary) ゲノム情報の基づき，超好熱菌に初めて見出したL-アスパラギン酸オキシダーゼが嫌気条件下でも機能する色素依存性L-アスパラギン酸脱水素酵素反応も触媒することを見出し，NAD生合成系の初発酵素として機能していることを提唱した．
32.	Toshihisa Ohshima and Haruhiko Sakuraba : L-Aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: Characteristics and role of the enzyme in the de novo biosynthesis of NAD proposed by genome sequencing, 11th German-Japanese Workshop on Enzyme Technology, Dresden, Oct. 2001.
33.	Toshihisa Ohshima and Haruhiko Sakuraba : A Novel ATP-formation system in sugar metabolism of the hyperthermophilic archaeon Pyrococcus furiosus, 2000 International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii, Dec. 2000. (Summary) 超好熱古細菌の解糖系に存在するADP依存性グルコキナーゼの遺伝子クローニング，AMPからATPを合成する新規キナーゼの発見，その酵素化学的諸性質および遺伝子の転写誘導についての知見を発表し，これまで不明であった超好熱古細菌のエネルギー代謝におけるこれらの酵素の役割を提唱した．
34.	Toshihisa Ohshima, Haruhiko Sakuraba, J. H. Schreier, Emi Utsumi and Naoki Nunoura : A novel site of glycolytic regulation in hyperthermophilic archaeon Pyrococcus furiosus: transcriptional regulation of amp-dependent (ATP-forming) kinase by maltose, The 3rd international congress on extremophiles, Hamburg, Germany, Sep. 2000. (Summary) AMPからATPを合成する新規キナーゼの発見，その酵素化学的諸性質および遺伝子の転写誘導についての知見を発表し，超好熱古細菌のエネルギー代謝において本酵素がATP生産に機能することを提唱した．
35.	Toshihisa Ohshima, Naoki Nunoura, Masayuki Fukuda, Nobuko Mori and Haruhiko Sakuraba : Extremely Thermostable Phenylalanine Dehydrogenase from Marine Thermophilic Baicillus Sp.:Characterization and Gene Cloning, Sequencing and Expression, The 2nd JSPS-NRCT Joint Seminar on Development of Thermotolerant Microbial Resources and Their Applications in Thailand and Japan, 131, Yamaguchi, Jan. 2000. (Summary) 高度耐熱性フェニルアラニン脱水素酵素が存在することを初めて見出し，これを単離精製し，その酵素化学的諸性質を明らかにした．また，遺伝子クローニングおよび大量発現系の構築に成功した
36.	Toshihisa Ohshima, Haruhiko Sakuraba, Emi Utsumi and Chizu Kujo : A novel bifunctional (AMP- andADP-dependent) pyruvate kinase is present in the hyperthermophilic Archaeon Pyrococcus furiosus, Thermophile'98, Brest, France, Sep. 1998. (Summary) 超好熱古細菌 Pyrococcus furiosus にAMP依存性のピルビン酸キナーゼが存在することを初めて見出し，これを単離精製し，その酵素化学的諸性質を明らかにした．また，超好熱古細菌のエネルギー代謝における本酵素の役割について考察した．
37.	Haruhiko Sakuraba and Toshihisa Ohshima : Characterization and application of dye-linked dehydrogenases from thermotolerant microorganisms :dye-linked L-malate dehydrogenase of thermotolerant Bacillus sp. DSM465, The 1st JSPS-NRCT Joint Seminar on Development of Thermotolerant Microbial Resources and Their Applications in Thailand and Japan, 122, Bangkok, Jan. 1998. (Summary) 好熱菌Bacillusに色素依存性リンゴ酸脱水素酵素が存在することを出し，これを単離精製し，その酵素化学的諸性質を明らかにした．本酵素は高度耐熱性であり応用面での有用性が明らかになった．

Proceeding of Domestic Conference:

1.	Ryushi Kawakami, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus furiosusのPLP依存性アミノ酸ラセマーゼの機能と構造の特徴, 2023年度日本ビタミン学会大会, Jun. 2023.
2.	Ryushi Kawakami, Tomoki Kawase, Taira Uehara, Haruhiko Sakuraba and Toshihisa Ohshima : PH0140転写因子によるアミノ酸ラセマーゼ遺伝子クラスターの発現制御, 2022年度極限環境生物学会, Nov. 2022.
3.	Ryushi Kawakami, Tomoki Kawase, Mikio Sato, Haruhiko Sakuraba and Toshihisa Ohshima : Mechanism of the expression of amino acid racemase gene controlled by L-Ile/ D-allo-Ile in the hyperthermophilic archaeon Pyrococcus horikoshii, 2021年度極限環境生物学会, Nov. 2021.
4.	Ryushi Kawakami, Nana Hanzawa, 大志田達也, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT-3のオルニチンアミノトランスフェラーゼの機能解析と構造解析, 2019年度日本ビタミン会大会, Jun. 2019.
5.	Ryushi Kawakami, Haruhiko Sakuraba, 大森勇門 and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT3のアミノ酸ラセマーゼの酵素化学的解析, 日本農芸化学会中四国支部第45回講演会, Jun. 2016.
6.	Ryushi Kawakami, 山下久美, 大森勇門, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT-3 由来アミノ酸ラセマーゼの同定, 2014年度日本ビタミン会大会, Jun. 2014.
7.	Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : L-プロリン脱水素酵素からのオキシダーゼ活性除去の検討, 2013年度日本生物工学会大会, Sep. 2013.
8.	山田洋平, Haruhiko Sakuraba, Toshihisa Ohshima, Yukako Hayashi, Tomoki Yabutani and Junko Motonaka : 高温環境下での安定性向上を指向した酵素固定化電極の試作と評価, 第68回日本分析化学討論会, May 2010.
9.	谷祐児, 糸山由希子, 西兼一, 山田洋平, 和田知賀博, 庄田佳生, 櫻庭春彦, Toshihisa Ohshima, Yukako Hayashi, Tomoki Yabutani and Junko Motonaka : 耐熱性D-プロリン脱水素酵素固定化カーボンナノチューブ-アガロース複合膜修飾電極の作製, 第55回ポーラログラフィーおよび電気分析化学討論会, Vol.55, No.3, 211, Nov. 2009.
10.	中島麻里, 田中勝人, 谷祐児, Tomoki Yabutani, Yukako Hayashi, 櫻庭春彦, Toshihisa Ohshima and Junko Motonaka : 耐熱性酵素を利用したアンペロメトリック分岐鎖アミノ酸フロー測定システムの開発, 2009年日本化学会西日本大会, Vol.1, 149, Nov. 2009.
11.	山田洋平, 和田知賀博, 遠藤剛, 渡邊敏夫, 谷祐児, Yukako Hayashi, Tomoki Yabutani, Junko Motonaka, 櫻庭春彦, Toshihisa Ohshima and 酒瀬川信一 : トリスオスミウム錯体/グルコースオキシターゼおよびビリルビンオキシターゼ/カーボンナノチューブゲル固定化電極の作製とそのバイオ電池への適用, 日本分析化学会第58年会, Vol.1, No.1, 77, Sep. 2009.
12.	中島麻里, 田中勝人, 谷祐児, Tomoki Yabutani, Yukako Hayashi, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 耐熱性酵素/電子メディエータ同時固定化電極を用いる分岐鎖アミノ酸フロー系センシングシステムの開発, 第15回分析化学若手セミナー, Aug. 2009.
13.	山田洋平, 和田知賀博, 谷裕児, Yukako Hayashi, Tomoki Yabutani, Junko Motonaka, 酒瀬川信一, Haruhiko Sakuraba and Toshihisa Ohshima : Bacillus Subtilis 由来 CotAのバイオセンサーへの利用, 2008年日本化学会西日本大会, 179, Nov. 2008.
14.	田中勝人, 谷祐児, Tomoki Yabutani, 三島有二, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 耐熱性ジアホラーゼ固定化電極の作製とそのNAD依存性酵素センサーへの応用, 第53回ポーラログラフィー及び電気分析化学討論会プログラム, Nov. 2007.
15.	田中勝人, 谷祐児, Tomoki Yabutani, 三島有二, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : NAD依存性酵素センシングのための耐熱性ジアホラーゼ固定化電極の作製とその応用, 日本分析化学会第56年会, Sep. 2007.
16.	末信一郎, 吉田達剛, 桶崎陽友, 鄭海涛, 米田一成, Haruhiko Sakuraba and Toshihisa Ohshima : 好熱菌由来L-リジン脱水素酵素を用いたL-リジンバイオセンシング, 第59回日本生物工学会大会, Sep. 2007.
17.	Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱菌由来アルドラーゼの結晶構造解析, 第59回日本生物工学会大会, Sep. 2007.
18.	清水泰博, 米田一成, 土居克己, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーケアSulfolobus tokodaiiのセリン生合成系の解明:ホスホグリセリンアミノ基転移酵素(PSAT)の機能解析, 日本農芸化学会中四国・西日本支部合同大会, Sep. 2007.
19.	谷祐児, Tomoki Yabutani, 三島有二, Shuichiro Goda, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 耐熱性D-プロリン脱水素酵素固定化電極の極性有機溶媒環境下における特性評価, 第68回分析化学討論会, May 2007.
20.	浅井一作, 米田一成, Toshihisa Ohshima and Haruhiko Sakuraba : 超好熱アーキアSulfolobus tokodaii strain7由来L-アスパラギン酸オキシダーゼのX線結晶構造解析, 日本農芸化学会中四国支部第18回講演会, May 2007.
21.	井手康裕, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱菌の耐熱性2-デオキシリボース-5-リン酸アルドラーゼ(DERA)の磁性化, 日本農芸化学会2007年度大会, Mar. 2007.
22.	黒田早織, Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshiiのNAD合成酵素(NADS)の機能解析, 日本農芸化学会2007年度大会, Mar. 2007.
23.	米田一成, Haruhiko Sakuraba, Hideaki Tsuge, 勝沼信彦 and Toshihisa Ohshima : 超好熱アーキアArchaeoglobus fulgidus由来L-アスパラギン酸脱水素酵素のX線結晶構造解析, 日本農芸化学会2007年度本大会, Mar. 2007.
24.	清水泰博, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアのセリン生合成系の解明:Sulfolobus tokodaii D-3-ホスホグリセリン酸脱水素酵素(PGDH)の機能解析, 日本農芸化学会2007年度大会, Mar. 2007.
25.	福田純也, 米田一成, 川上竜巳, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii由来L-リジン脱水素酵素ホモログ遺伝子PH1688の機能解析, 日本農芸化学会2007年度大会, Mar. 2007.
26.	田中勝人, 谷祐児, Tomoki Yabutani, 三島有二, Shuichiro Goda, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 耐熱性NAD依存性酵素/ジアホラーゼ電極の作製とその特性評価, 2006年日本化学会西日本大会, Nov. 2006.
27.	Toshihisa Ohshima, 米田一成, Haruhiko Sakuraba, Hideaki Tsuge and 勝沼信彦 : 超好熱アーキアArchaeoglobus fulgidus由来のデノボNAD生合成系の初発酵素L-アスパラギン酸脱水素酵素の機能と構造解析, 第406回ビタミンB研究委員会, Nov. 2006.
28.	Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : 低温菌由来の巨大グルタミン酸脱水素酵素, 酵素工学研究会, Oct. 2006.
29.	谷祐児, Tomoki Yabutani, 三島有二, Shuichiro Goda, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 耐熱性D-プロリン脱水素酵素-寒天固定化電極の作製とその特性評価, 日本分析化学会第55年会, Sep. 2006.
30.	Toshihisa Ohshima, 里村武範, Haruhiko Sakuraba, 谷祐児, Tomoki Yabutani and Junko Motonaka : 超好熱菌の膜結合性D-プロリン脱水素酵素の機能開発, 酵素・補酵素を楽しむ会, Sep. 2006.
31.	清水泰博, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱好酸性アーキアSulfolobus tokodaii strain 7 由来D-3-ホスホグリセリン酸脱水素酵素(PGDH)の機能解析, 日本生物工学会大会, Sep. 2006.
32.	Toshihisa Ohshima, 里村武範, Ryushi Kawakami, Haruhiko Sakuraba, 谷裕児, Tomoki Yabutani and Junko Motonaka : 超好熱アーキアPyrobaculum islandicumの色素依存性D-プロリン脱水素酵素の機能解析と応用, 第2回D-アミノ酸研究学術講演会, Sep. 2006.
33.	吉原久美子, 高橋克幸, Toshihisa Ohshima and Haruhiko Sakuraba : 超好熱菌Thermotoga maritima及びPyrobaculum aerophilum由来2-deoxy-D-ribose-5-phosphate aldolase(DERA)が触媒するアルドール縮合反応, 日本農芸化学会中四国支部大会, Sep. 2006.
34.	清水泰博, 吉田晴美, Shuichiro Goda, Haruhiko Sakuraba, 柊弓弦 and Toshihisa Ohshima : 超好熱アーケアAeropyrum pernixK1由来5'-deoxy-5'-methylthioadenosinephosphorylase(MTAP)の機能解析, 日本Archaea研究会第19回講演会, Aug. 2006.
35.	米田一成, Haruhiko Sakuraba, Hideaki Tsuge, 勝沼信彦 and Toshihisa Ohshima : 超好熱アーキアArchaeoglobus fulgidus由来L-アスパラギン酸脱水素酵素のX線結晶構造解析, 日本ビタミン学会第58回大会, May 2006.
36.	Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 低温菌Janthinobacterium lividum UTB1302が生産するNAD依存性グルタミン酸脱水素酵素の遺伝子クローニング, 日本ビタミン学会第58回大会, May 2006.
37.	Shuichiro Goda, 西川良美, Haruhiko Sakuraba, 小島正樹, 柊弓絃 and Toshihisa Ohshima : 大腸菌内で不活性な構造で生産される超好熱アーキア Pyrobaculum islandicum 由来グルタミン酸脱水素酵素の活性化機構の解明, 日本ビタミン学会第58回大会, May 2006.
38.	吉行貴司, 伊藤佳洋, Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアSulfolobus tokodaii strain7 由来2-keto-3-deoxygluconate (KDG) aldolase の機能解析, 日本農芸化学会2006年度大会, Mar. 2006.
39.	寺井利光, 広瀬由鷹, 末信一郎, 堀照夫, Ryushi Kawakami, Haruhiko Sakuraba and Toshihisa Ohshima : 耐熱性L-プロリン脱水素酵素の電極上への分子配向制御固定, 日本農芸化学会2006年度大会, Mar. 2006.
40.	桃沢茂行, 桃沢茂行, 清水泰博, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : :Pyrococcus horikoshii OT-3由来，Quinolinate phosphoriboosyltransferaseの諸性質の解明, 日本農芸化学会関西支部第442回講演会, Dec. 2005.
41.	谷祐児, Tomoki Yabutani, 三島有二, Shuichiro Goda, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 高温溶解性ゲルを用いた耐熱性膜酵素電極の構築とその特性評価, 第51回ポーラログラフィーおよび電気分析化学討論会, Nov. 2005.
42.	Toshihisa Ohshima : 生かせ，微生物のポテンシャル, 2005年度日本農芸化学会関西·中四国·西日本合同大会シンポジウム, Sep. 2005.
43.	Ryushi Kawakami, Haruhiko Sakuraba, Hideaki Tsuge, 勝沼信彦 and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshiiの新規色素依存性L-プロリン脱水素酵素の機能と構造, 日本Archaea研究会第18回講演会, Jul. 2005.
44.	Ryushi Kawakami, 田頭祐哉, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアArchaeoglobus fulgidusのNAD生合成に機能すると予想されるL-AspDHホモログの機能解析, 日本ビタミン学会第57回大会, May 2005.
45.	Haruhiko Sakuraba, 米田一成, Toshihisa Ohshima, Hideaki Tsuge and 勝沼信彦 : 超好熱アーキアPyrococcus horikoshii由来キノリン酸シンターゼのX線結晶構造解析, 日本ビタミン学会第57回大会, May 2005.
46.	Toshihisa Ohshima : 超好熱アーキアのヌクレオチド補欠因子要求性酵素の研究, 日本ビタミン学会第57回大会ビタミン学会賞受賞講演, May 2005.
47.	谷祐児, Tomoki Yabutani, 三島有二, Shuichiro Goda, Haruhiko Sakuraba, Toshihisa Ohshima and Junko Motonaka : 耐熱性D-プロリン脱水素酵素固定化電極の作製とその電気化学特性評価, 第66回分析化学討論会, May 2005.
48.	Toshihisa Ohshima, 本多恭子, Ryushi Kawakami, Haruhiko Sakuraba and 新居修 : スクモ藍建て工程に関与するインジゴ還元酵素の精製と性質, 日本農芸化学会2005年度大会, Mar. 2005.
49.	Haruhiko Sakuraba, Ryushi Kawakami, Hideaki Tsuge, Shuichiro Goda, 勝沼信彦, 安藝謙嗣 and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshiiに存在する新規ATP含有アミノ酸脱水素酵素, 日本農芸化学会2005年度大会, Mar. 2005.
50.	米田一成, Haruhiko Sakuraba, Hideaki Tsuge, 勝沼信彦, 倉光成紀, 川端猛 and Toshihisa Ohshima : 超好熱アーキアSulforobus tokodaii由来機能未知タンパク質ST1625のX線結晶構造解析, 日本農芸化学会2005年度大会, Mar. 2005.
51.	Shuichiro Goda, Yoshimi Nishikawa, Masaki Kojima, Haruhiko Sakuraba, Yuzuru Hiragi and Toshihisa Ohshima : X線小角散乱法を用いた超好熱アーキア由来リコンビナント酵素の活性化による構造変化の解明, 日本農芸化学会2005年度大会, Mar. 2005.
52.	廣瀬由鷹, 木村友和, 鄭海涛, 末信一朗, 堀照夫, 片山秀夫, Ryushi Kawakami and Toshihisa Ohshima : リコンビナント耐熱性L-プロリン脱水素酵素を用いたLegionella属菌のDNA検出システム, 日本農芸化学会2005年度大会, Mar. 2005.
53.	Toshihisa Ohshima : 超好熱アーキアのL-プロリン脱水素酵素複合体の機能·構造解析と応用, 21世紀COEプログラム「微生物機能の戦略的活用による生産基盤拠点」第4回シンポジウム, Mar. 2005.
54.	Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshiiの新規プロリン脱水素酵素:補欠分子族の同定と立体構造解析, 第399回ビタミンB研究協議会, Feb. 2005.
55.	Hideaki Tsuge, Ryushi Kawakami, Haruhiko Sakuraba, 吾郷日出夫, 宮野雅司, 安藝謙嗣, 勝沼信彦 and Toshihisa Ohshima : P. horikoshii由来L-プロリン脱水素酵素のX線結晶構造解析, 日本生物物理学会第42回年会, Dec. 2004.
56.	西川良美, Shuichiro Goda, 米田一成, Haruhiko Sakuraba, Hideaki Tsuge, 勝沼信彦, 柊弓絃 and Toshihisa Ohshima : 大腸菌で生産されるPyrobaculum islandicum由来のグルタミン酸脱水素酵素の活性化機構, 日本生物物理学会第42回年会, Dec. 2004.
57.	Toshihisa Ohshima, Ryushi Kawakami, Haruhiko Sakuraba and Hideaki Tsuge : 超好熱アーキアの色素依存性脱水素酵素の探索とその応用, 第27回日本分子生物学会年会, 369, Dec. 2004.
58.	Toshihisa Ohshima, 田頭祐哉, Shuichiro Goda and Haruhiko Sakuraba : 超好熱アーキアArchaeoglobus fulgidusのNAD(P)依存性L-アスパラギン酸脱水素酵素ホモログ遺伝子の機能解析, 第4回極限環境微生物学会年会, Vol.3, No.2, 45-46, Nov. 2004.
59.	Toshihisa Ohshima : 超好熱菌のアルドラーゼの機能開発, 第77回日本生化学会大会ランチョンセミナー, Vol.76, No.8, 1145, Oct. 2004.
60.	Yaruhiro Shimizu, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : Cloning, Expression, and Characterization of L-Threonine Dehydrogenase from a Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3, 第77回日本生化学会大会, Vol.76, No.8, 825, Oct. 2004.
61.	Yoshihisa Suzuki, Gen Sazaki, Yoshiki Matsuura, Hironori Hondoh, Haruhiko Sakuraba, Toshihisa Ohshima, Kazuo Nakajima and Katsuhiro Tamura : 高圧力下におけるグルコースイソメラーゼ結晶の成長促進機構に関する分子論的考察, 第45回高圧討論会, Oct. 2004.
62.	廣瀬由鷹, 木村友和, 鄭海涛, 末信一朗, 堀照夫, 片山秀夫, Ryushi Kawakami and Toshihisa Ohshima : 超好熱アーキア由来色素依存性L-プロリン脱水素酵素を用いたLegionella属菌検出DNAセンサーの構築, 日本生物工学会平成16年度大会, 240, Sep. 2004.
63.	Haitao Zheng, Yukata Hirose, Tomokazu Kimura, Shin-ichiro Suye, Teruo Hori, Hideo Katayama, Ryushi Kawakami and Toshihisa Ohshima : Multilayer assembly of dye-linked L-proline dehydrogenase and poly(allylamine)ferrocene by layer-by layer adsorption method, 日本生物工学会平成16年度大会, 240, Sep. 2004.
64.	Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 低温菌Janthinobacterium lividum UTB1302由来のグルタミン酸脱水素酵素の特徴, 日本生物工学会平成16年度大会, 109, Sep. 2004.
65.	川内由香, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 土壌由来環境DNAからのアラニン脱水素酵素遺伝子の検索, 日本生物工学会平成16年度大会, 57, Sep. 2004.
66.	田頭祐哉, Toshihisa Ohshima, Haruhiko Sakuraba and Shuichiro Goda : 超好熱性アーキアArchaeoglobus fulgidusのL-アスパラギン酸脱水素酵素ホモログ(AF1838)の機能解析, 日本農芸化学会2004年度中四国支部大会, Sep. 2004.
67.	Yoshihisa Suzuki, Gen Sazaki, Yoshiki Matsuura, Hironori Hondoh, Haruhiko Sakuraba, Toshihisa Ohshima, Kazuo Nakajima and Katsuhiro Tamura : A molecular-level discussion on the growth kinetics of glucose isomerase crystals, 第34回結晶成長国内会議, Aug. 2004.
68.	Toshihisa Ohshima : 超好熱菌由来のNAD依存性L-リジン脱水素酵素, ビタミンB研究委員会第397回夏期研究協議会, Vol.78, No.9, 453-454, Aug. 2004.
69.	Hideyuki Nakagawa, Takefumi Hosotani, Kuniko Nagasaka, Toshinori Nagasaka, Haruhiko Sakuraba and Toshihisa Ohshima : Isolation of a novel lectin from the dorsal venom of Japanese devil scorpionfish, Inimicus japonicus, 生化学, Vol.76, No.8, 1012, Aug. 2004. (Summary) 広島産オニオコゼの背鰭毒棘のタンパク質画分より，レクチンの精製を試みた．毒棘標品をアフィニティークロマトグラフィーにより分画·精製を行い，Dーマンノース結合性レクチンを分離した． (Keyword) Fish venom / Inimicus japonicus / Lectin / Dorsal spines / Mitogenic activity
70.	Toshihisa Ohshima, Haruhiko Sakuraba, Mohammad W Bhuiya, Shuichiro Goda, Hideaki Tsuge and 勝沼信彦 : 超好熱アーキアPyrobaculum islandicumのNAD依存性グルタミン酸脱水素酵素の構造と機能相関, 日本Archaea研究会第17回講演会, Jul. 2004.
71.	Ryushi Kawakami, Hideaki Tsuge, Shuichiro Goda, Haruhiko Sakuraba, 勝沼信彦, 安藝謙嗣 and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT-3から発見されたフラビン酵素，色素依存性L-プロリン脱水素酵素の機能と構造解析, 日本ビタミン学会第56回大会, Vol.78, No.4, 253, May 2004.
72.	Haruhiko Sakuraba, Ryushi Kawakami and Toshihisa Ohshima : 超好熱アーキアThermococcus litoralisに存在する新規アラニン:グリオキシル酸アミノトランスフェラーゼ, 日本ビタミン学会第56回大会, Vol.78, No.4, 246, May 2004.
73.	Toshihisa Ohshima, Haruhiko Sakuraba, Hideaki Tsuge and 勝沼信彦 : 超好熱菌Pyrobaculum islandicumのNAD依存性グルタミン酸脱水素酵素の構造解析, 第396回ビタミンB研究委員会, Vol.78, No.7, 368-369, May 2004.
74.	Shuichiro Goda, Yoshimi Nishikawa, Haruhiko Sakuraba, Yuzuru Hiragi and Toshihisa Ohshima : 大腸菌で発現された超好熱アーキア由来酵素の活性化機構の解明, 日本生化学会中国四国支部大会, May 2004.
75.	Toshihisa Ohshima, Haruhiko Sakuraba and Shuichiro Goda : 高機能エンザイム創製へのニューアプローチ, 日本農芸化学会2004年度大会, 429, Mar. 2004.
76.	Shuichiro Goda, Haruhiko Sakuraba, Yutaka Kawarabayasi and Toshihisa Ohshima : 超好熱アーキア由来アグマチナーゼの大腸菌を用いた発現系の構築とその酵素機能の解析, 日本農芸化学会2004年度大会, 263, Mar. 2004.
77.	本多恭子, Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba, 新居修 and Toshihisa Ohshima : 藍染め染色液中の好アルカリ性菌の分離とインジゴ還元酵素の諸性質の解明, 日本農芸化学会2004年度大会, 263, Mar. 2004.
78.	西川良美, Shuichiro Goda, Mohammad W Bhuiya, Haruhiko Sakuraba, 柊弓絃 and Toshihisa Ohshima : 大腸菌で発現される超好熱アーキア由来のグルタミン酸脱水素酵素の活性化機構, 日本農芸化学会2004年度大会, 118, Mar. 2004.
79.	Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba, Hideaki Tsuge and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT-3の色素依存性L-プロリン脱水素酵素の性質と構造, 日本農芸化学会2004年度大会, 118, Mar. 2004.
80.	米田一成, Haruhiko Sakuraba, Shuichiro Goda, Hideaki Tsuge, 勝沼信彦 and Toshihisa Ohshima : 超好熱アーキアMethanococcus janaschii由来bifunctional ADP依存性Glucokinase/PhosphofructokinaseのX線結晶構造解析, 日本農芸化学会2004年度大会, 98, Mar. 2004.
81.	Toshihisa Ohshima, 伊藤佳洋, Haruhiko Sakuraba, Shuichiro Goda and Yutaka Kawarabayasi : 超好熱アーキアSulfolobus tokodaiiの新規NAD(P)依存性グルコース脱水素酵素の機能解析, 極限環境微生物学会2003年度年会, Dec. 2003.
82.	Toshihisa Ohshima : 好気·好酸性超好熱アーキアSulfolobus tokodaiiの新規NAD(P)依存性グルコース脱水素酵素の機能解析, ビタミンB研究委員会第394回研究評議会, Vol.78, No.1, 60-61, Nov. 2003.
83.	Haruhiko Sakuraba, Kazunari Yoneda, Hideaki Tsuge, Nobuhiko Katunuma, Shuichiro Goda and Toshihisa Ohshima : Crystallization and preliminary X-ray diffraction analysis of ADP-dependent glucokinase/phosphofructokinase, a bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii, 第76回日本生化学会大会, Vol.75, No.8, 1079, Oct. 2003.
84.	Haruhiko Sakuraba, Ikuko Shimoya, Hideaki Tsuge, Shuichiro Goda, Nobuhiko Katunuma and Toshihisa Ohshima : Unique Tetrameric Structure of 2-Deoxyribose-5-Phosphate Aldorase from The Hyperthermophilic Archaea Aeropyrum pernix, 第76回日本生化学会大会, Vol.75, No.8, 1079, Oct. 2003.
85.	Shuichiro Goda, Koji Ono, Ryushi Kawakami, Haruhiko Sakuraba, Yutaka Kawarabayasi and Toshihisa Ohshima : Pyrococcus horikoshii agmatinase: gene expression in E. coli and characterization, 第76回日本生化学会大会, Vol.75, No.8, 1066, Oct. 2003.
86.	Mojgan Heydari, Toshihisa Ohshima, Naoki Kominato and Haruhiko Sakuraba : Highly Stable Lysine Dehydrogenase from Geobacillus stearothermophilus: Characterization, Gene Cloning, Sequencing and Expression, 日本生物工学会平成15年度大会, 46, Sep. 2003.
87.	Mohammad W Bhuiya, Hideaki Tsuge, Takahito Imagawa, Nobuhiko Katunuma, Haruhiko Sakuraba and Toshihisa Ohshima : Crystallization and preliminary X-ray diffraction analysis of L-aspartate oxidase from the hyperthermophilic archaeon, Pyrococcus furiosus, 日本生物工学会平成15年度大会, 48, Sep. 2003.
88.	Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT-3に存在する2種類の色素依存性L-プロリン脱水素酵素の特徴, 日本生物工学会平成15年度大会, 45, Sep. 2003.
89.	Toshihisa Ohshima : 古細菌研究から見えてくる生命初期進化の道筋，遺伝子進化と水平伝播, 日本進化学会第5回大会, 62, Aug. 2003.
90.	Toshihisa Ohshima, Haruhiko Sakuraba, 下家郁子, Shuichiro Goda, Hideaki Tsuge and 勝沼信彦 : 超好熱アーキアAeropyrum pernixの2-デオキシ-D-リボース-5-リン酸アルドラーゼの機能と立体構造解析, 第3回日本蛋白質科学会年会, 106, Jun. 2003.
91.	Shuichiro Goda, Haruhiko Sakuraba, Mohammad W Bhuiya, 里村武範, Ryushi Kawakami, 倉光成紀, 柊弓絃 and Toshihisa Ohshima : 大腸菌によって発現された低活性超好熱菌由来グルタミン酸脱水素酵素の熱及び尿素による活性化過程のX線小角散乱と熱量計による解析, 日本アーキア研究会第16回講演会, 26, Jun. 2003.
92.	Toshihisa Ohshima, Haruhiko Sakuraba, 金井匡平, Shuichiro Goda and Yutaka Kawarabayasi : 超好熱アーキアのゲノム情報に基づくNMN-アデニルトランスフェラーゼの機能解析, 日本ビタミン学会第55回大会, May 2003.
93.	Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアThermococcus profundusの色素依存性L-プロリン/NADH脱水素酵素複合体の解析, 日本農芸化学会2003年度大会, 258, Apr. 2003.
94.	Shuichiro Goda, Haruhiko Sakuraba, Mohammad W Bhuiya, 里村武範, Ryushi Kawakami, 柊弓絃 and Toshihisa Ohshima : 好熱菌由来グルタミン酸脱水素酵素の熱及び尿素による成熟化課程の物理化学的解明, 日本農芸化学会2003年度大会, 241, Apr. 2003.
95.	三谷有理, Haruhiko Sakuraba, Shuichiro Goda, Yutaka Kawarabayasi and Toshihisa Ohshima : 超高熱アーキアで最初のATP依存性グルコキナーゼ:Aeropyrum pernix酵素の発現系の構築及び諸性質, 日本農芸化学会2003年度大会, 100, Apr. 2003.
96.	Haruhiko Sakuraba, 下家郁子, Hideaki Tsuge, 勝沼信彦, Shuichiro Goda and Toshihisa Ohshima : 超好熱アーキアAeropyrum pernix由来2-Deoxyribose-5-phosphate Aldolaseの結晶構造解析, 日本農芸化学会2003年度大会, 100, Apr. 2003.
97.	Hideyuki Nakagawa, Fumihiko Satoh, Hiroko Kagota, Koji Hiraga, Kuniko Nagasaka, Toshinori Nagasaka, Haruhiko Sakuraba and Toshihisa Ohshima : Isolation of a cytotoxic factor from the dorsal spines of the redfin velvetfish, Hypodytes rubripinnis, Journal of Pharmacological Sciences, Vol.91, 130, Mar. 2003. (Summary) 小型刺毒魚であるハオコゼの背鰭毒棘のタンパク質画分より，細胞障害性を示す糖タンパク質をアフィニティークロマトグラフィーにより精製した，Native PAGE では分子量11万であった．SDS-PAGE では7.6 万，3.0 万，1 万数千の位置にそれぞれ，タンパク質バンドを呈した．7.6 万のサブユニットのN末端アミノ酸はアスパラギン酸であり，12残基のアスパラギン酸まで分析された． (Keyword) Hypodytes rubripinnis / Fish venom / Glycoprotein / Cytotoxic protein / Dorsal spine
98.	Toshihisa Ohshima and Haruhiko Sakuraba : 極限環境生態系からの有用遺伝子資源探索, 第25回日本分子生物学会年会, 416, Dec. 2002.
99.	田村隆明, Haruhiko Sakuraba, Toshihisa Ohshima and 新居修 : 藍染め染色剤(スクモ)の生産に関与する微生物群の解析, 第3回極限環境微生物学会年会, 82-83, Dec. 2002.
100.	三谷有理, Shuichiro Goda, Haruhiko Sakuraba, Yutaka Kawarabayasi and Toshihisa Ohshima : 好気性超好熱アーキアAeropyrum pernix由来のATP依存性グルコキナーゼ:発現系の構築及び諸性質の解明, 第3回極限環境微生物学会年会, 67-68, Dec. 2002.
101.	里村武範, Ryushi Kawakami, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアD-アミノ酸脱水素酵素の性質, 第3回極限環境微生物学会年会, 5-6, Dec. 2002.
102.	Toshihisa Ohshima and Haruhiko Sakuraba : ポストゲノム時代における新しい生物工学を目指して, 日本生物工学会平成14年度大会, Oct. 2002.
103.	里村武範, Ryushi Kawakami, 藤崎礼子, 中山奈央子, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 好熱好酸性アーキアSulfolobus tokodaii strain 7 由来 D- 乳酸脱水素酵素の特徴, 日本生物工学会平成14年度大会, Oct. 2002.
104.	Ryushi Kawakami, 里村武範, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT3 の色素依存性 L- プロリン脱水素酵素の精製と酵素遺伝子の同定, 日本生物工学会平成14年度大会, Oct. 2002.
105.	Mojgan Heydari, T Nakahira, Haruhiko Sakuraba, Junko Motonaka and Toshihisa Ohshima : A novel electrocatalytic biosensor using dye-linked L-proline dehydrogenase from a Hyperthermophile, 日本生物工学会平成14年度大会, Oct. 2002.
106.	蒲生原新太郎, Haruhiko Sakuraba and Toshihisa Ohshima : 嫌気性超好熱アーキアPyrococcus horikoshii OT3 の酸化ストレス応答タンパク質の特徴, 第75回日本生化学会大会, Vol.74, No.8, 1074, Oct. 2002.
107.	下家郁子, Yutaka Kawarabayasi, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキア由来2-デオキリシボース-5-リン酸アルドラーゼの大量発現系の構築及び諸性質の解明, 第75回日本生化学会大会, Vol.74, No.8, 1001, Oct. 2002.
108.	久次米旭, 柊弓絃, Hideaki Tsuge, 市村薫, Shuichiro Goda, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus horikoshii OT3 の ATP 依存性グルコキナーゼの構造特性, 第75回日本生化学会大会, Vol.74, No.8, 994, Oct. 2002.
109.	Mohammad W Bhuiya, Haruhiko Sakuraba, Hideaki Tsuge, Nobuhiko Katunuma and Toshihisa Ohshima : Crystallization and preliminary X-ray diffraction analysis of glutamate dehydrogenase from the hyperthermophilic archaeon, Pyrobaculum islandicum, 第75回日本生化学会大会, Vol.74, No.8, 989, Oct. 2002.
110.	Fumihiko Satoh, Hideyuki Nakagawa, Haruo Yamada, Kuniko Nagasaka, Toshinori Nagasaka, Haruhiko Sakuraba and Toshihisa Ohshima : 刺毒魚ハオコゼの毒棘糖タンパク質の精製, 第75回日本生化学会大会, Vol.74, No.8, 727, Oct. 2002. (Summary) ハオコゼの背ビレ毒棘から糖タンパク質を精製した． (Keyword) redfin velvetfish / lectin / cytotoxicity / glycoprotein
111.	Toshihisa Ohshima, Ryushi Kawakami and Haruhiko Sakuraba : 超好熱アーキアの色素依存性L-プロリン脱水素酵素の特徴, 第388回ビタミンB研究委員会, Aug. 2002.
112.	Toshihisa Ohshima, Haruhiko Sakuraba and 辻真奈美 : 超好熱アーキアのグルタミン酸脱水素酵素の部位特異的変異による高度耐熱性ノルバリン脱水素酵素の創製, 日本アーキア研究会第15回講演会, Jul. 2002.
113.	伊藤佳洋, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアSulfolobus tokodaiiのNAD(P)-グルコース脱水素酵素遺伝子の機能解析, 日本ビタミン学会第54回大会, Apr. 2002.
114.	Toshihisa Ohshima, 金沢勇, Haruhiko Sakuraba, Yutaka Kawarabayasi and 菊池久 : ゲノム情報に基づく超好熱アーキアPyrococcus horikoshiiのNADキナーゼの機能解析, 第387回ビタミンB研究委員会, Vol.76, No.5, 318, Apr. 2002.
115.	Haruhiko Sakuraba, 芳陵一生, 古賀晋治, 高橋守 and Toshihisa Ohshima : ADP依存性グルコキナーゼ/ホスホフルクトキナーゼ，Methanococcus jannaschiiに存在する新規二機能性酵素, 日本農芸化学会2002年度大会, 137, Mar. 2002.
116.	金井匡平, Toshihisa Ohshima, Haruhiko Sakuraba, Yutaka Kawarabayasi and 菊池久 : 超好熱アーキアPyrococcus horikoshii OT-3 のゲノム情報に基づくNADP生合成系の解明:NMN-アデニリルトランスフェラーゼの機能解析, 日本農芸化学会2002年度大会, 117, Mar. 2002.
117.	金澤勇, Haruhiko Sakuraba, Toshihisa Ohshima, Yutaka Kawarabayasi and 菊池久 : 超好熱アーキアPyrococcus horikoshii OT-3 のゲノム情報に基づくNADP生合成の解明:NADキナーゼの機能解析, 日本農芸化学会2002年度大会, 117, Mar. 2002.
118.	竹原英毅, 酒瀬川信一, 牟田滋, Haruhiko Sakuraba, Toshihisa Ohshima and 久原哲 : Flavobacterium meningosepticum由来グリセロールキナーゼの安定化の分子機構, 日本農芸化学会2002年度大会, 95, Mar. 2002.
119.	酒瀬川信一, 竹原英毅, 芳陵一生, Haruhiko Sakuraba and Toshihisa Ohshima : Flavobacterium meningosepticum由来グリセロールキナーゼの安定化, 日本農芸化学会2002年度大会, 95, Mar. 2002.

Et cetera, Workshop:

1.	Toshihisa Ohshima, Haruhiko Sakuraba, Ryushi Kawakami and Hideaki Tsuge : 超好熱菌のアミノ酸代謝関連酵素，NAD生合成系酵素の立体構造解析, タンパク3000プロジェクト代謝系評価会, Nov. 2006.
2.	Toshihisa Ohshima : 次世代型酵素電極バイオセンサーチップの開発, 徳島大学テクノクラスター基調講演, Jun. 2004.
3.	関口順一, 青山紘一, Toshihisa Ohshima, 大島淳, 河原秀久, 清水和幸, 西村基弘, 播磨武 and 室岡義勝 : 世界をリードする生物工学教育とは, Seibutsu-kogaku Kaishi, Vol.79, No.12, 470-489, Dec. 2001.
4.	Toshihisa Ohshima : 特殊環境微生物を利用した阿波藍加工システム構築の可能性試験, 文部科学省地域研究開発促進拠点支援事業<新技術説明会 in 徳島>, 33-41, Jul. 2001.
5.	Toshihisa Ohshima : 2000環太平洋国際化学会議日本化学会国際大会，Pacifichem2000, バイオサイエンスとインダストリー, Vol.59, No.2, 114, Jan. 2001.
6.	Toshihisa Ohshima and Haruhiko Sakuraba : 高効率蛋白質発現システムの開発(超好熱菌のタンパク質折り畳み機構を利用した高効率蛋白質発現システムの開発), 平成11年度知的基盤創造·利用技術研究開発事業報告書, 65-99, Jan. 2000.
7.	Toshihisa Ohshima : 超好熱菌のピルビン酸キナーゼの構造と機能に関する研究, サッポロ生物科学振興財団第14回研究報告書, 7-11, Jan. 2000.
8.	Toshihisa Ohshima and Haruhiko Sakuraba : 新規超好熱用培養装置の開発と超好熱菌の分離, 三島海雲記念財団研究報告書(平成10年度), Vol.36, 11-14, Jan. 1999.

Report:

1.	Toshihisa Ohshima : Studies on biosynthesis of coenzymes, NAD(P) in hyperthermophilic archaea, --- Functional and structural analysis of the related enzymes based on genome information ---, Reports of Reserch Assisted by The Asahi Glass Foundation, 41-42, Tokyo, Dec. 2004.
2.	Toshihisa Ohshima, Junko Motonaka, Shuichiro Goda and 和田友良 : 新規酵素バイオセンサーの開発, 徳島大学地域共同研究センター研究成果報告書, Vol.13, 68-69, Tokushima, Aug. 2004.
3.	Toshihisa Ohshima, Haruhiko Sakuraba and 高橋正明 : アフィニティリガンド結合磁性粒子の開発, 徳島大学地域共同研究センター研究成果報告書, Vol.13, 31-32, Tokushima, Aug. 2004.
4.	Toshihisa Ohshima : 超好熱菌の酸化還元酵素の機能と構造解析及び機能創製, バイオテクノロジー先端技術シーズ培養研究平成15年度成果報告集, 113-130, Mar. 2004.
5.	Ri-ichi Murakami, Toshihisa Ohshima, Kiyoshi Fukui, Hideyuki Nakagawa, Haruhiko Sakuraba and Daisuke Yonekura : ダイヤモンドなど炭素系薄膜を利用した新規高度耐久性バイオ基材の開発, 文部科学省科学研究費補助金(基盤研究(C)(2))成果報告書, Mar. 2004.
6.	Toshihisa Ohshima : 超好熱菌の酸化還元酵素の機能と構造解析及び機能創製, バイオテクノロジー先端技術シーズ培養研究平成14年度成果報告集, 69-80, Mar. 2003.
7.	Toshihisa Ohshima : 超好熱菌の酸化還元酵素の機能と構造解析及び機能創製, バイオテクノロジー先端技術シーズ培養研究平成13年度成果報告集, 75-95, Mar. 2002.
8.	Toshihisa Ohshima : 超好熱菌の酸化還元酵素の機能と構造解析及び機能創製, バイオテクノロジー先端技術シーズ培養研究平成12年度成果報告集, 71-95, Mar. 2001.

Patent:

1.	Toshihisa Ohshima, Haruhiko Sakuraba and Shuichiro Goda : 新規なアスパラギン酸デヒドロゲナーゼ, 2005-073757 (Mar. 2005), .
2.	Toshihisa Ohshima, 本多恭子 and 新居修 : インジゴ還元酵素及びその製造方法, 2004-248034 (Aug. 2004), .
3.	Toshihisa Ohshima, 田島秀二 and 高橋正明 : 反応性色素結合磁性粒子及び蛋白質分離精製法, 2004-99303 (Mar. 2004), .
4.	松本和也, 数野康, 東村紀一, Toshihisa Ohshima and Haruhiko Sakuraba : キラルなヒドロキシアルデヒド化合物の製造方法, 2004-95263 (Mar. 2004), .
5.	Toshihisa Ohshima, Haruhiko Sakuraba and Shuichiro Goda : アグマチナーゼおよびその製造方法, 2003-283447 (Jul. 2003), 2005-046102 (Feb. 2005), .
6.	Toshihisa Ohshima, Haruhiko Sakuraba, Ryushi Kawakami, 吉田洋一 and 下家郁子 : 耐熱性2-デオキシリボース-5-リン酸アルドラーゼ, 2002-049172 (Feb. 2002), 2003-250553 (Sep. 2003), .
7.	Naoki Nunoura, 石川宏美, Haruhiko Sakuraba and Toshihisa Ohshima : 耐熱性リシン脱水素酵素，その遺伝子，それを含有する組換えベクター及びそれを含有する形質転換体並びにそれを用いた耐熱性リシン脱水素酵素の製造方法, 2001-261402 (Aug. 2001), 2003-61681 (Mar. 2003), .
8.	Naoki Nunoura, 森修子, 福田雅之, 公庄千寿, Haruhiko Sakuraba and Toshihisa Ohshima : 耐熱性フェニルアラニン脱水素酵素遺伝子，それを含有する組換えベクター，その組換えベクターを含有する形質転換体及びその形質転換体を用いた耐熱性フェニルアラニン脱水素酵素の製造方法, 2000-261170 (Aug. 2000), 2002-065270 (Mar. 2002), .
9.	Toshihisa Ohshima, 福田雅之, 森修子, Naoki Nunoura and Haruhiko Sakuraba : L-フェニルアラニン脱水素酵素及びその製造法, 2000-030519 (Feb. 2000), 2001-218579 (Aug. 2001), .

Professor Emeritus : Ohshima, Toshihisa

Research

Field of Study

Subject of Study

Book / Paper

Book:

Academic Paper (Judged Full Paper):

Academic Paper (Unrefereed Paper):

Review, Commentary:

Proceeding of International Conference:

Proceeding of Domestic Conference:

Et cetera, Workshop:

Report:

Patent: