1994 Fiscal Year Annual Research Report

C-C結合形成反応を触媒する耐熱性酵素による生理活性物質の新規合成法の開発

Research Project

Project/Area Number	05558080
Research Institution	TOHOKU UNIVERSITY
Principal Investigator	小倉協三東北大学, 反応化学研究所, 教授 (80006303)
Co-Investigator(Kenkyū-buntansha)	小畑充生トヨタ自動車(株), 研究部バイオラボ, 主任研究員古山種俊東北大学, 工学部, 助教授 (20089808)
Keywords	プレニルトランスフェラーゼ / ファルネシル二リン酸 / 耐熱性酵素 / 部位特異的変異 / 人工基質ホモログ / 合成酵素 / 基質特異性 / ゲラニル二リン酸
Research Abstract	中等度好熱性細菌B.stearothermophilusのファルネシル二リン酸(FPP)合成酵素については(1)部位特異的変異の導入(2)耐熱性変異型FPP合成酵素の大腸菌内での大量産生(3)熱処理と2段階のカラムクロマトグラフィーによる精製の系を確立した。この系を用いてプレニルトランスフェラーゼに特徴的な7つの保存領域内で特によく保存されているアミノ酸について変異型酵素を作成し、それらの触媒機能の変化を精査し、新規なC-C結合形成反応の触媒機能獲得の有無を調べた。領域VIIに保存されているArg-295をValに置換した変異型酵素R295Vは酵素活性はほとんど変化しなったが、非アルカリ性基質イソペンテニルニリン酸(IPP)に対するKm値が野生型のFPP合成酵素のそれの約1.5倍に増大した。同様の変化がC末端のHisをLeuに換えたH297Lでも認められた。この変化はプライマー基質をジメチルアリル二リン酸(DMAPP)にした場合さらに顕著になり、変異体のKm値は約3倍となった。さらにCys-289をPheに換えたC289Fでは10倍になった。領域VIに保存されているモチーフDDXXDのAspの変異体、D224A、D224E、D225I、D228Aはいずれも触媒活性が激減したが、反応連度論的解析により、基質に対するKm値はIPP、GPPのいずれに対しても大きな変化はなかった。D288Aのみが例外で、IPPに対するKm値が野生型の10倍にもなった。領域VIの下流に保存されているLys-238の変異体酵素K238AおよびK238Rはいずれも触媒活性には大きな変化はなかったが、IPPに対するKm値がそれぞれ4.2倍5.1倍になった。これらの変異体酵素はいずれもIPPに対するKm値が増大しているのでホモアリル性の基質に対する特異性が特に変化している可能性がある。今後これらの変異体酵素の人工基質ホモログに対する基質特異性を精査して、野生型酵素では得られない新規なC-C結合形成反応の触媒となる変異体酵素を見出す実験を展開して行く予定である。

Research Products
(26 results)

All Other

All Publications (26 results)

[Publications] T.Koyama,et al: "Thermostable Farnesyl Diphosphate Synthase of Bacillus stearothermophilus:Molecular Cloning,Sequence Determination,Overproductio,and Purification" J.Biochem.113. 355-363 (1993)
[Publications] H.Sagami,et al: "Formation of Dolichol from Dehydrodolichol is Catalyzed by NADPH-dependent Reductase Localized in Microsomes of Rat Liver" J.Biol.Chem.,. 268. 10109-10113 (1993)
[Publications] H.Sagami,et al: "Biosynthesis of Prenyl Diphosphates by Cell-free Extracts from Mammalian Tissues" J.Biochem.114. 112-117 (1993)
[Publications] H.Sagami,et al: "Geranylgeranyl Diphosphate Synthase Catalyzing the Single Condensation between Isopentenyl Diphosphate and Faernesyl Diphosphate" J.Biochem.114. 118-121 (1993)
[Publications] S.Ohnuma,et al: "Alteration of Product Specificties of Prenyltransferases by Metal lons" Biochem.Biophys.Res.Commun.192. 407-412 (1993)
[Publications] H.Inoue,et al: "Formation of Farnesal and 3-Hydroxy-2,3-dihydrofamesal from Farnesol by Protoplasts of Botryococcus Braunii" Biochem.Biophys.Res.Commun.196. 1401-1405 (1993)
[Publications] H.Nakane,et al: "Crystallization and Preliminary X-ray Diffraction Studies of Bacillus Stearothermophilus.Farnesyl Diphosphate Synthase Expressed in Escherichia coli" J.Mol.Biol.233. 787-788 (1993)
[Publications] S.Ohnuma,et al: "Solanesyl Diphosphate Synthase Reaction with Artificial Substrartes.Formation of R and S-Enatiomers of 4-and8-Methyl Derivatives of Geranylgeranyl Diphosphate" Bioorg.Med.Chem.Lett.3. 2733-2738 (1993)
[Publications] K.Saiki,et al: "In vitro Heme O Synthesis by the cyoE Gene Product from Escherchia coli" J.Biol.Chem.26. 26041-26043 (1993)
[Publications] H.Sagami,et al: "Purfication and Properties of Geranylgeranyl Diphosphate Synthase from Bovine Brain." J.Biol.Chem.269. 20561-20566 (1994)
[Publications] H.Inoue,et al: "Puosphoryration of Farnesol by a Cell-Free System from Botryococcus Braunii" Biochem.Biophys.Res.Commun.200. 1036-1041 (1994)
[Publications] H.Inoue,et al: "Formation of Farnesyl Oleate and Three Other Farnesyl Fatty Acid Esters by Cell-free Extracts from Botryococcus Braunii B Race" Phytochemistry. 36. 1203-1207 (1994)
[Publications] T.Koyama,et al: "Sie-directed Mutagenesis of Farnesyl Diphosphate Synthase:Effect of Substitution on the Three Carboxyl Terminal Amino Acids" Can.J.Chem.72. 75-79 (1994)
[Publications] T.Koyama,et al: "Thermostable Farnesyl Diphosphate Synthase of Bacillus stearothermophilus:Crystallization and Site-directed Mutagenesis" Acta Biochim.Polonica.41. 281-292 (1994)
[Publications] H.Sagami,et al: "Purification of Geranylgeranyl Diphoshate from Bovine Brain" Acta Biochim.Polonica.41. 283-302 (1994)
[Publications] Y.Maki,et al: "Farnesyl Diphosphate Synthase Reacitions of Geranyl Diphosphate Analoguse Having Oxygen Atoms in Their Alkyl chains" Chem.Lett.10. 1841-1844 (1994)
[Publications] T.Koyama,et al: "Structural and Functional Roles of the Cysteine Residues of Bacillus stearothermophikus Farnesyl Diphosphate Synyhase" Biochemistry. 33. 12644-12648 (1994)
[Publications] H.Sagami,et al: "Novel Isoprenoid Modified Proteins in Halobacteria" Biochem.Biophys.Res Commun.203. 972-976 (1994)
[Publications] T.Koyama,et al: "Isopen teny Diphospghate Isomerase in Rubber Latex" Phytochemisty. (In Press).
[Publications] K.Asawatreratanakul,rt al.: "Characterization of Hevea Isopentenyl Diphosphate Isomerase" Proceedings of the 11th FAOBMB Symposium. (In Press).
[Publications] T.Koyama,et al.: "Analysis of Prenyltransferase Reaction Products from C-Serum of Hevea brasiliensis" Proceedings of the 11th FAOBMB Symposium. (In Press).
[Publications] H.Inoue,et al.: "Properties of Farnesol Phosphokinase of Botryococcus braunii" Phytochemistry. (In Press).
[Publications] 佐上博、他: "ブレニル化蛋白質の発見とオソプレノイド生化学の新展開" 生化学. 12. 1488-1501 (1994)
[Publications] 小倉協三: "酵素研究の新展開(-島英治他偏):イソプレノイド生化学の新展開" 東京化学同人(印刷予定),
[Publications] 小倉協三: "有機化学の最近の展開(日本化学研究会編):イノプレノイドの生合成化学" 培風館(印刷予定),
[Publications] 古山種俊: "新タンパク質応用工学第11章第4節" フジテクノシステムズ(株),(印刷予定),

1994 Fiscal Year Annual Research Report

C-C結合形成反応を触媒する耐熱性酵素による生理活性物質の新規合成法の開発

Principal Investigator

小倉 協三 東北大学, 反応化学研究所, 教授 (80006303)

Research Products

[Publications] T.Koyama,et al: "Thermostable Farnesyl Diphosphate Synthase of Bacillus stearothermophilus:Molecular Cloning,Sequence Determination,Overproductio,and Purification" J.Biochem.113. 355-363 (1993)

[Publications] H.Sagami,et al: "Formation of Dolichol from Dehydrodolichol is Catalyzed by NADPH-dependent Reductase Localized in Microsomes of Rat Liver" J.Biol.Chem.,. 268. 10109-10113 (1993)

[Publications] H.Sagami,et al: "Biosynthesis of Prenyl Diphosphates by Cell-free Extracts from Mammalian Tissues" J.Biochem.114. 112-117 (1993)

[Publications] H.Sagami,et al: "Geranylgeranyl Diphosphate Synthase Catalyzing the Single Condensation between Isopentenyl Diphosphate and Faernesyl Diphosphate" J.Biochem.114. 118-121 (1993)

[Publications] S.Ohnuma,et al: "Alteration of Product Specificties of Prenyltransferases by Metal lons" Biochem.Biophys.Res.Commun.192. 407-412 (1993)

[Publications] H.Inoue,et al: "Formation of Farnesal and 3-Hydroxy-2,3-dihydrofamesal from Farnesol by Protoplasts of Botryococcus Braunii" Biochem.Biophys.Res.Commun.196. 1401-1405 (1993)

[Publications] H.Nakane,et al: "Crystallization and Preliminary X-ray Diffraction Studies of Bacillus Stearothermophilus.Farnesyl Diphosphate Synthase Expressed in Escherichia coli" J.Mol.Biol.233. 787-788 (1993)

[Publications] S.Ohnuma,et al: "Solanesyl Diphosphate Synthase Reaction with Artificial Substrartes.Formation of R and S-Enatiomers of 4-and8-Methyl Derivatives of Geranylgeranyl Diphosphate" Bioorg.Med.Chem.Lett.3. 2733-2738 (1993)

[Publications] K.Saiki,et al: "In vitro Heme O Synthesis by the cyoE Gene Product from Escherchia coli" J.Biol.Chem.26. 26041-26043 (1993)

[Publications] H.Sagami,et al: "Purfication and Properties of Geranylgeranyl Diphosphate Synthase from Bovine Brain." J.Biol.Chem.269. 20561-20566 (1994)

[Publications] H.Inoue,et al: "Puosphoryration of Farnesol by a Cell-Free System from Botryococcus Braunii" Biochem.Biophys.Res.Commun.200. 1036-1041 (1994)

[Publications] H.Inoue,et al: "Formation of Farnesyl Oleate and Three Other Farnesyl Fatty Acid Esters by Cell-free Extracts from Botryococcus Braunii B Race" Phytochemistry. 36. 1203-1207 (1994)

[Publications] T.Koyama,et al: "Sie-directed Mutagenesis of Farnesyl Diphosphate Synthase:Effect of Substitution on the Three Carboxyl Terminal Amino Acids" Can.J.Chem.72. 75-79 (1994)

[Publications] T.Koyama,et al: "Thermostable Farnesyl Diphosphate Synthase of Bacillus stearothermophilus:Crystallization and Site-directed Mutagenesis" Acta Biochim.Polonica.41. 281-292 (1994)

[Publications] H.Sagami,et al: "Purification of Geranylgeranyl Diphoshate from Bovine Brain" Acta Biochim.Polonica.41. 283-302 (1994)

[Publications] Y.Maki,et al: "Farnesyl Diphosphate Synthase Reacitions of Geranyl Diphosphate Analoguse Having Oxygen Atoms in Their Alkyl chains" Chem.Lett.10. 1841-1844 (1994)

[Publications] T.Koyama,et al: "Structural and Functional Roles of the Cysteine Residues of Bacillus stearothermophikus Farnesyl Diphosphate Synyhase" Biochemistry. 33. 12644-12648 (1994)

[Publications] H.Sagami,et al: "Novel Isoprenoid Modified Proteins in Halobacteria" Biochem.Biophys.Res Commun.203. 972-976 (1994)

[Publications] T.Koyama,et al: "Isopen teny Diphospghate Isomerase in Rubber Latex" Phytochemisty. (In Press).

[Publications] K.Asawatreratanakul,rt al.: "Characterization of Hevea Isopentenyl Diphosphate Isomerase" Proceedings of the 11th FAOBMB Symposium. (In Press).

[Publications] T.Koyama,et al.: "Analysis of Prenyltransferase Reaction Products from C-Serum of Hevea brasiliensis" Proceedings of the 11th FAOBMB Symposium. (In Press).

[Publications] H.Inoue,et al.: "Properties of Farnesol Phosphokinase of Botryococcus braunii" Phytochemistry. (In Press).

[Publications] 佐上 博、他: "ブレニル化蛋白質の発見とオソプレノイド生化学の新展開" 生化学. 12. 1488-1501 (1994)

[Publications] 小倉 協三: "酵素研究の新展開(-島英治他偏):イソプレノイド生化学の新展開" 東京化学同人(印刷予定),

[Publications] 小倉 協三: "有機化学の最近の展開(日本化学研究会編):イノプレノイドの生合成化学" 培風館(印刷予定),

[Publications] 古山 種俊: "新タンパク質応用工学第11章第4節" フジテクノシステムズ(株),(印刷予定),

小倉協三東北大学, 反応化学研究所, 教授 (80006303)

[Publications] 佐上博、他: "ブレニル化蛋白質の発見とオソプレノイド生化学の新展開" 生化学. 12. 1488-1501 (1994)

[Publications] 小倉協三: "酵素研究の新展開(-島英治他偏):イソプレノイド生化学の新展開" 東京化学同人(印刷予定),

[Publications] 小倉協三: "有機化学の最近の展開(日本化学研究会編):イノプレノイドの生合成化学" 培風館(印刷予定),

[Publications] 古山種俊: "新タンパク質応用工学第11章第4節" フジテクノシステムズ(株),(印刷予定),