1996 Fiscal Year Annual Research Report

イソプレイド生合成における鎖延長酵素反応機構の解明

Research Project

Project/Area Number	06403033
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)	佐上博東北大学, 反応化学研究所, 助教授 (10134058) 古山種俊東北大学, 工学部, 助教授 (20089808)
Keywords	イソプレノイド / 生合成 / ファルネシル二リン酸合成酵素 / ゲラニルゲラニル二リン酸合成酵素 / プレニルトランスフェラーゼ / ヘプタプレニル二リン酸合成酵素 / イソペンテニル二リン酸合成酵素 / テルペノイド
Research Abstract	1.Bacilus stearothermophilusの遺伝子クローニングにより得た耐熱性のファルネシルニリン酸合成酵素につき、種々の部位特異的変異体を作成し、それらの活性変動を調べた。その結果、すべてのプレニルトランスフェラーゼに共通して存在するFQXXDD配列中のFQすなわち220位と221位のフェニルアラニンとグルタミンの残基は、ともに触媒機能に必須の役割をもつことが明らかになった。また、リシン-47とリシン-183がともにホモアリル基質IPPの結合に重要な役割を果たすことを明らかにした。これらの結果を総合して、本酵素の反応機構解明に深いメスをいれることができた。 2.B. stearothermophilusのヘプタプレニルニリン酸合成酵素を構成する二つの異なるタンパク質の遺伝子クローニングに成功し、それらの塩基配列を決定した。一方のタンパク質は既知のプレニルトランスフェラーゼと一次構造上有意義な相同性を示すが、他方のタンパク質は既知の如何なるタンパク質とも類似性が認められない。しかし、この2つのタンパク質が存在しないと酵素機能を発現しないことが証明された。この2つの遺伝子は機能不明のタンパク質の遺伝子を狭んでDNA上に位置していたが、このタンパク質はメナキノン生合成の最終段階であるプレニルナフトキノンのメチル化酵素であることを証明した。さらに、B. subtilisのヘプタプレニルニリン酸を構成する2つのタンパク質の遺伝子クローニングおよび大量発現にも成功した。 3.ウシ大脳由来のゲラニルゲラニルニリン酸合成酵素の完全精製を達成した。また、この酸素は高等植物の酸素とは異なり、ジメチルアリルニリン酸とイソペンテニルニリン酸との縮合反応を触媒しないことを明らかにした。さらにこの酸素は、ゲラニルゲラニルニリン酸による生成物阻害を受けることを明らかにした。 4.古細菌Halobacterium halobiumにメバロン酸を投与して、タンパク質画分への取り組みを追求した。その結果新しいタイプのイソプレノイド修飾タンパク質を見いだし、このタンパク質のシステインはジフィタニルグリセリル基で修飾されていることを明らかにした。 5.緑藻Botryococcus brauniiにファルネソ-ルのリン酸化酵素が存在することを見いだし、その酵素学的諸性質を明らかにした。 6.酵母中にドリコールとデヒドロドリコールをそれぞれ対応するアルデヒドに酸化する代謝系が存在することを明らかにした。

Research Products

(31 results)

All Other

All Publications (31 results)

[Publications] H. Sagami at al: "Purification and Properties of Geranylgeranyl Diphoshate Synthase from Bovine Brain" J. Biol. Chem. 269. 20561-20566 (1994)
[Publications] H. Inoue et al: "Phosphorylation 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" Plrytochemistry. 36. 1203-1207 (1994)
[Publications] T. Koyama et al: "Site-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 Disphoshate Synthase of Bacillus Stearothermophilus : Crystallization and Site-directed Mutagenesis" Acta Biochim. Polonica.41. 281-292 (1994)
[Publications] H. Sagami et al.: "Purifation of Geranylgeranyl Diphoshate from Bovine Brain" Acta Biochim. Polonica.41. 293-302 (1994)
[Publications] Y. Maki et al: "Farnesyl Diphoshate Synthase Reactions of Geranyl Diphosphate Analogues Having Oxygen Atoms in Their Alkyl Chains" Chem. Lett.1841-1844 (1994)
[Publications] T. Koyama et al: "Structural and Functional Roles of the Cysteine Residues of Bacillus stearothermophilus Farnesyl Disphoshase Synthase" Biochemistry. 33. 12644-12648 (1994)
[Publications] H. Sagami et al: "Novel Isoprenoid Modified Proteins in Halobacteria" Biochem. Biophys. Res. Commum.203. 072-976 (1994)
[Publications] Y. Maki et al.: "Farnesyl Diphoshate Synthase Reaction with Dimethylallyl Diphoshate Analogues Having Oxygen Atoms in Their Chains" Chem. Lett. 389-390 (1995)
[Publications] H. Sagami et al.: "A Novel Type of Protein Modification by Isoprenoid-derived Materials" J. Biol. Chem.270. 14851-14854 (1995)
[Publications] A. Koike-Takeshita et al.: "Molecular Cloning and Nucleotide Sequences of the Genes for Two Essential Proteins Constituting a Novel Enzyme System for Heptaprenyl Diphoshate Synthesis" J. Biol. Chem.270. 18396-18400 (1995)
[Publications] T. Koyama et al.: "Significance of Phe-220 Gin-221 in the Catalytic Mechanism of farnesyl Diphosphate Synthase of Bacillus stearothermophilus" Biochem. Biophys. Res. Commun.212. 681-686 (1995)
[Publications] Y. Maki et al: "Substrate Specificity of Farnesyl Diphosphate Synthase from Bacillus stearothermophilus" Bioorg. Med. Chem. Lett.5. 1605-1608 (1995)
[Publications] H. Inoue et al.: "Properties of Farnesol Phosphokinase of Botryococcus braunii" Phytochemistry. 40. 377-381 (1995)
[Publications] H. Sagami et al: "Enzymatic Formation of Dehydrodolichal and Dolichal, New Products Related to Yeast Dolichol Biosynthesis" J. Biol. Chem. 271. 9560-9566 (1996)
[Publications] T. Koyama et al.: "Identification of Significant Residues in the Substrate Binding Site of Farnesyl Diphosphate Synthase" Biochemistry. 35. 9533-9538 (1996)
[Publications] S. Ohnuma et al.: "Conversion from Farnesyl Diphoshate Synthase to Geranylgeranyl Diphoshate Synthase by Random Chamical Mutagenesis" J. Biol. Chem.271. 10087-10095 (1996)
[Publications] A. Kurisaki et al.: "Proteolytic Release of Dehydrodolichyl Diphoshate Synthase from Pig Testis Microsome" Biosci. Biotech. Biochem.60. 1109-1114 (1996)
[Publications] T. Koyama et al.: "Isopentenyl Diphosphate Isomerase in Rubber Latex" Phytochemistry. 43. 769-772 (1996)
[Publications] A. Kurisaki et al.: "Distribution of Polyprenols and Dolichols in Soybean Plant" Phytochemistry. 44. 45-50 (1997)
[Publications] A. Koike-Takeshita et al.: "Identification of Novel Gene Cluster Participating in Menaquinone (Vitamin K_2) Biosynthesis. Clorung and Sequence Determination of the 2-Heptaprenyl-1, 4-naphthoquinone Methyltransferase Gene of Bacillus stearothermophilus" J. Biol. Chem.(in press).
[Publications] Y. Zhan et al.: "Two Cistrons of the gerC Operon of Bacillus subtilis Encode the Two Subunits Heptaprenyl Diphoshate Symthase" J. Bacteriol.(in press).
[Publications] T. Ohkura et al.: "A Partial Defeciency of Dehydrodolichol Reduction is a Cause Carbohydrate-Defecient Glycoprotein Syndrome Type I"
[Publications] J. Biol. Chem. (in press).
[Publications] 佐上博、小倉協三: "プレニル化蛋白質の発見とイソプレノイド生化学の新展開" 生化学, 14 (1994)
[Publications] 小倉協三: "酵素研究の新展開(一島英治他編):イソプレノイド成合成の分子機構" 東京化学同人, 12 (1995)
[Publications] 小倉協三: "有機化学のフロンティア(日本化学研究会編):イソプレノイドの成合成" 培風館, 22 (1995)
[Publications] 小倉協三: "酵素による炭素・炭素結合形成反応と有機合成への応用" 有機合成化学会誌, 11 (1996)
[Publications] K. Ogura, T. Koyama: "Mechanistic Enzymology and Molecular Genetics of Chain Elongation in Isoprenoid Biosynthesis" Kodansya Scientific, 23 (1996)
[Publications] K. Ogura: "Polyprenyl Diphoshate Synthases" Plenum Publishing Corporation (in press),

1996 Fiscal Year Annual Research Report

イソプレイド生合成における鎖延長酵素反応機構の解明

Principal Investigator

小倉 協三 東北大学, 反応化学研究所, 教授 (80006303)

Research Products

[Publications] H. Sagami at al: "Purification and Properties of Geranylgeranyl Diphoshate Synthase from Bovine Brain" J. Biol. Chem. 269. 20561-20566 (1994)

[Publications] H. Inoue et al: "Phosphorylation 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" Plrytochemistry. 36. 1203-1207 (1994)

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

[Publications] H. Sagami et al.: "Purifation of Geranylgeranyl Diphoshate from Bovine Brain" Acta Biochim. Polonica.41. 293-302 (1994)

[Publications] Y. Maki et al: "Farnesyl Diphoshate Synthase Reactions of Geranyl Diphosphate Analogues Having Oxygen Atoms in Their Alkyl Chains" Chem. Lett.1841-1844 (1994)

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

[Publications] H. Sagami et al: "Novel Isoprenoid Modified Proteins in Halobacteria" Biochem. Biophys. Res. Commum.203. 072-976 (1994)

[Publications] Y. Maki et al.: "Farnesyl Diphoshate Synthase Reaction with Dimethylallyl Diphoshate Analogues Having Oxygen Atoms in Their Chains" Chem. Lett. 389-390 (1995)

[Publications] H. Sagami et al.: "A Novel Type of Protein Modification by Isoprenoid-derived Materials" J. Biol. Chem.270. 14851-14854 (1995)

[Publications] A. Koike-Takeshita et al.: "Molecular Cloning and Nucleotide Sequences of the Genes for Two Essential Proteins Constituting a Novel Enzyme System for Heptaprenyl Diphoshate Synthesis" J. Biol. Chem.270. 18396-18400 (1995)

[Publications] T. Koyama et al.: "Significance of Phe-220 Gin-221 in the Catalytic Mechanism of farnesyl Diphosphate Synthase of Bacillus stearothermophilus" Biochem. Biophys. Res. Commun.212. 681-686 (1995)

[Publications] Y. Maki et al: "Substrate Specificity of Farnesyl Diphosphate Synthase from Bacillus stearothermophilus" Bioorg. Med. Chem. Lett.5. 1605-1608 (1995)

[Publications] H. Inoue et al.: "Properties of Farnesol Phosphokinase of Botryococcus braunii" Phytochemistry. 40. 377-381 (1995)

[Publications] H. Sagami et al: "Enzymatic Formation of Dehydrodolichal and Dolichal, New Products Related to Yeast Dolichol Biosynthesis" J. Biol. Chem. 271. 9560-9566 (1996)

[Publications] T. Koyama et al.: "Identification of Significant Residues in the Substrate Binding Site of Farnesyl Diphosphate Synthase" Biochemistry. 35. 9533-9538 (1996)

[Publications] S. Ohnuma et al.: "Conversion from Farnesyl Diphoshate Synthase to Geranylgeranyl Diphoshate Synthase by Random Chamical Mutagenesis" J. Biol. Chem.271. 10087-10095 (1996)

[Publications] A. Kurisaki et al.: "Proteolytic Release of Dehydrodolichyl Diphoshate Synthase from Pig Testis Microsome" Biosci. Biotech. Biochem.60. 1109-1114 (1996)

[Publications] T. Koyama et al.: "Isopentenyl Diphosphate Isomerase in Rubber Latex" Phytochemistry. 43. 769-772 (1996)

[Publications] A. Kurisaki et al.: "Distribution of Polyprenols and Dolichols in Soybean Plant" Phytochemistry. 44. 45-50 (1997)

[Publications] A. Koike-Takeshita et al.: "Identification of Novel Gene Cluster Participating in Menaquinone (Vitamin K_2) Biosynthesis. Clorung and Sequence Determination of the 2-Heptaprenyl-1, 4-naphthoquinone Methyltransferase Gene of Bacillus stearothermophilus" J. Biol. Chem.(in press).

[Publications] Y. Zhan et al.: "Two Cistrons of the gerC Operon of Bacillus subtilis Encode the Two Subunits Heptaprenyl Diphoshate Symthase" J. Bacteriol.(in press).

[Publications] T. Ohkura et al.: "A Partial Defeciency of Dehydrodolichol Reduction is a Cause Carbohydrate-Defecient Glycoprotein Syndrome Type I"

[Publications] J. Biol. Chem. (in press).

[Publications] 佐上 博、小倉協三: "プレニル化蛋白質の発見とイソプレノイド生化学の新展開" 生化学, 14 (1994)

[Publications] 小倉協三: "酵素研究の新展開(一島英治他編):イソプレノイド成合成の分子機構" 東京化学同人, 12 (1995)

[Publications] 小倉協三: "有機化学のフロンティア(日本化学研究会編):イソプレノイドの成合成" 培風館, 22 (1995)

[Publications] 小倉協三: "酵素による炭素・炭素結合形成反応と有機合成への応用" 有機合成化学会誌, 11 (1996)

[Publications] K. Ogura, T. Koyama: "Mechanistic Enzymology and Molecular Genetics of Chain Elongation in Isoprenoid Biosynthesis" Kodansya Scientific, 23 (1996)

[Publications] K. Ogura: "Polyprenyl Diphoshate Synthases" Plenum Publishing Corporation (in press),

小倉協三東北大学, 反応化学研究所, 教授 (80006303)

[Publications] 佐上博、小倉協三: "プレニル化蛋白質の発見とイソプレノイド生化学の新展開" 生化学, 14 (1994)