1991 Fiscal Year Annual Research Report

基質と酵素間の精密構造認識と反応機構

Research Project

Project/Area Number	03236103
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)	左右田健次京都大学, 化学研究所, 教授 (30027023) 中嶋暉躬東京大学, 薬学部, 教授 (50012597) 三川潮東京大学, 薬学部, 教授 (60012613)
Keywords	イソプレノイド / ファルネシル二リン酸合成酵素 / カルコン合成酵素 / イソフラボン / マストパラン / カルモジュリン / Dーアミノ酸トランスアミナ-ゼ / キメラタンパク質
Research Abstract	小倉らは中等度好熱性細菌のファルネシル二リン酸合成酵素の遺伝子をクロ-ニングし、その塩基配列を調べた結果、リボソ-ム結合部位につづいて、GTGで始まり297個のアミノ酸をコ-ドするOpen reading frameを形成していることがわかった。アミノ酸配列は対応する大腸菌の酵素のそれと40%の相同性があり、また、ヒト、ラットおよび酵母のそれらとの間に共通性の高い3箇所のドメインが認められた。さらに、この好熱性菌のファルネシル二リン酸合成酵素遺伝子が大腸菌で大量に発現できるプラスミドを構築することができた。三川らはクズ培養細胞を用いてイソフラボノイド生合成を研究し、カルコン合成酵素の遺伝子クロ-ニングと大腸菌での発現に成功し、Phaseolus由来の酵素との間に高い相同性のあることを確認した。また、イソフラボン合成に関与するPー450の可溶化と再構成に成功した。中嶋らはハチ毒ペプチドであるマストパランのすべての残基がD配座のアミノ酸から成る対掌体マストパランがマストパランと同様にカルモジュリンと結合し、競合的にカルモジュリン活性を阻害することを見いだしたが、この対掌体の結合部位はマストパランのそれとは異なることを明らかにした。また、カルモジュリンのカルシウム結合特性への影響もマストパランとその対掌体とでは異なることを見いだし、カルモジュリン分子にいろいろなペプチド鎖認識部位が存在する可能性を示した。左右田らは相同性の高いDーアミノ酸トランスアミナ-ゼと分岐鎖Lーアミノ酸トランスアミナ-ゼの遺伝子を融合させてキメラタンパク質を発現させ、それを均一に精製することに成功した。また、このキメラタンパク質は触媒をもたないが、補酵素ピリドキサルリン酸を結合する機能をもつことを明らかにした。

Research Products
(22 results)

All Other

All Publications (22 results)

[Publications] Ohnuma,S.: "Purification of Solanesyl Diphosphate Synthase from Micrococcus liteus.A New Class of Prenyltraltransferase" J.Biol.Chem.266. 22706-22713 (1991)
[Publications] Sagami,H.: "Formation of Z,E,E-Geranylgeranyl Diphosphate by Rat Liver Microsomes" J.Biol.Chem.266. 3458-3463 (1991)
[Publications] Matsuoka,S.: "Variable Product Specificity of Microsomal Dehydrodolichyl Diphosphate Synthase from Rat Liver" J.Biol.Chem.266. 3463-468 (1991)
[Publications] Koyama,T.: "Enzymatic Synthesis of Glycinoprenols" Tetrahedron Lett.32. 241-242 (1991)
[Publications] 古山種俊: "イソプレノイド鎖延長酵素の機能" 日本農芸化学会誌. 65. 1791-1793 (1991)
[Publications] Gotoh,T.: "Farnesyl Diphosphate Synthase and Solanesyl Diphosphate Synthase Reactions of Diphosphateーmodified Allylic Analogues" J.Biochem.(1992)
[Publications] Piek,T.: "Pharmacological Characterization and Chemical Fractionation of the Venoma of the Ponerine Ant,Paraponera clavata (F.)" Comp.Biochem.Physiol.99C. 481-486 (1991)
[Publications] Hagiwara,K.: "Determination of Biogeneic Aminesin Spider Venom Grands of Nine Typical Japanese Species and Chromatographic Elution Pattern Analysis" Jpn.J.Sanit.Zool.42. 77-84 (1991)
[Publications] Teshima,T.: "Total Synthesis of NSTX-3,Spider Toxin of Nephila Maculata" Tetrahedron. 47. 3305-3312 (1991)
[Publications] Kawai,N.: "Spider Toxin and the Glutamate Receptors" Comp.Biochem.Physiol.98C. 87-95 (1991)
[Publications] Koike,M.: "Chemotactic Peptide from Ropalidian Wasp as well as the Authentic Chemotactic Tripeptide Stimulates Two Distinct Pathways in Neutrophils,but the [Lys7] Analog Does Only one of Them" Biochem.Biophys.Res.Commun.175. 165-172 (1991)
[Publications] Ichinose,K.: "Regioselectivity of Biomimetic Reduction of Tetrahydronapth thalene.Location of Transition Structures for Hydride Addition with Theoretical Calculation" Chemistry Letters. 219-222 (1991)
[Publications] Hakamatsuka,T.: "Induced Isofalvonoids from Copper Chloride-Treated Stems of Pueralia lobata" Phytochemistry. 30. 1481-1482 (1991)
[Publications] Nakajima,O.: "Isolation.Scquence and Bacterial Expression of a cDNA for Chalcone Synthase from the Cultured Cells of Pueralia lobata" Chem.Pharm.Bull.39. 1911-1913 (1991)
[Publications] Hakamatsuka,T.: "P-450-Dependent Oxidative Rearrangement in Isoflavone Biosynthesis:Reconstitution of P-450 and NADPH:P-450 Reductase" Tetrahedron. 47. 5969-5978 (1991)
[Publications] Song,Y.: "Identification of plant Factors Inducing Virlence Gene Expression of Agrobactcrium tumcfaciens" Chem.Pharm.Bull.39. 2347-2350 (1991)
[Publications] Nishimura,K.: "Effect of Substitution of a Lysyl Residue That Binds Pyridoxal Phosphate in Thermostable D-Amino Acid Aminotransferase by Arginine and Alanine" Biochemistry. 30-16. 4072-4077 (1991)
[Publications] Yagi,T.: "Characterization of the Half Overall Reactions Catalyzed by L-Lysine:2-Oxoglutarate 6-Aminotransferase" J.Biochem.109-1. 61-65 (1991)
[Publications] Sung,M.: "Thermostable Aspatrate Aminotransferase from a Thermophilic Bacillus sp." J.Biol.Chem.266-4. 2567-2572 (1991)
[Publications] Toyama,H.: "Thermostable Alanine Recemase of Bacillus stearothermophilus:Subunit Dissociation and Unfolding" J.Biochem.110-2. 279-283 (1991)
[Publications] Toyama,H.: "Thermostable Alanine Racemase of Bacillus stearothermophilus" J.Biol.Chem.266-21. 13634-13639 (1991)
[Publications] 山田秀明: "微生物の機能開発" 学会出版センタ-, 326 (1992)

1991 Fiscal Year Annual Research Report

基質と酵素間の精密構造認識と反応機構

Principal Investigator

小倉 協三 東北大学, 反応化学研究所, 教授 (80006303)

Research Products

[Publications] Ohnuma,S.: "Purification of Solanesyl Diphosphate Synthase from Micrococcus liteus.A New Class of Prenyltraltransferase" J.Biol.Chem.266. 22706-22713 (1991)

[Publications] Sagami,H.: "Formation of Z,E,E-Geranylgeranyl Diphosphate by Rat Liver Microsomes" J.Biol.Chem.266. 3458-3463 (1991)

[Publications] Matsuoka,S.: "Variable Product Specificity of Microsomal Dehydrodolichyl Diphosphate Synthase from Rat Liver" J.Biol.Chem.266. 3463-468 (1991)

[Publications] Koyama,T.: "Enzymatic Synthesis of Glycinoprenols" Tetrahedron Lett.32. 241-242 (1991)

[Publications] 古山 種俊: "イソプレノイド鎖延長酵素の機能" 日本農芸化学会誌. 65. 1791-1793 (1991)

[Publications] Gotoh,T.: "Farnesyl Diphosphate Synthase and Solanesyl Diphosphate Synthase Reactions of Diphosphateーmodified Allylic Analogues" J.Biochem.(1992)

[Publications] Piek,T.: "Pharmacological Characterization and Chemical Fractionation of the Venoma of the Ponerine Ant,Paraponera clavata (F.)" Comp.Biochem.Physiol.99C. 481-486 (1991)

[Publications] Hagiwara,K.: "Determination of Biogeneic Aminesin Spider Venom Grands of Nine Typical Japanese Species and Chromatographic Elution Pattern Analysis" Jpn.J.Sanit.Zool.42. 77-84 (1991)

[Publications] Teshima,T.: "Total Synthesis of NSTX-3,Spider Toxin of Nephila Maculata" Tetrahedron. 47. 3305-3312 (1991)

[Publications] Kawai,N.: "Spider Toxin and the Glutamate Receptors" Comp.Biochem.Physiol.98C. 87-95 (1991)

[Publications] Koike,M.: "Chemotactic Peptide from Ropalidian Wasp as well as the Authentic Chemotactic Tripeptide Stimulates Two Distinct Pathways in Neutrophils,but the [Lys7] Analog Does Only one of Them" Biochem.Biophys.Res.Commun.175. 165-172 (1991)

[Publications] Ichinose,K.: "Regioselectivity of Biomimetic Reduction of Tetrahydronapth thalene.Location of Transition Structures for Hydride Addition with Theoretical Calculation" Chemistry Letters. 219-222 (1991)

[Publications] Hakamatsuka,T.: "Induced Isofalvonoids from Copper Chloride-Treated Stems of Pueralia lobata" Phytochemistry. 30. 1481-1482 (1991)

[Publications] Nakajima,O.: "Isolation.Scquence and Bacterial Expression of a cDNA for Chalcone Synthase from the Cultured Cells of Pueralia lobata" Chem.Pharm.Bull.39. 1911-1913 (1991)

[Publications] Hakamatsuka,T.: "P-450-Dependent Oxidative Rearrangement in Isoflavone Biosynthesis:Reconstitution of P-450 and NADPH:P-450 Reductase" Tetrahedron. 47. 5969-5978 (1991)

[Publications] Song,Y.: "Identification of plant Factors Inducing Virlence Gene Expression of Agrobactcrium tumcfaciens" Chem.Pharm.Bull.39. 2347-2350 (1991)

[Publications] Nishimura,K.: "Effect of Substitution of a Lysyl Residue That Binds Pyridoxal Phosphate in Thermostable D-Amino Acid Aminotransferase by Arginine and Alanine" Biochemistry. 30-16. 4072-4077 (1991)

[Publications] Yagi,T.: "Characterization of the Half Overall Reactions Catalyzed by L-Lysine:2-Oxoglutarate 6-Aminotransferase" J.Biochem.109-1. 61-65 (1991)

[Publications] Sung,M.: "Thermostable Aspatrate Aminotransferase from a Thermophilic Bacillus sp." J.Biol.Chem.266-4. 2567-2572 (1991)

[Publications] Toyama,H.: "Thermostable Alanine Recemase of Bacillus stearothermophilus:Subunit Dissociation and Unfolding" J.Biochem.110-2. 279-283 (1991)

[Publications] Toyama,H.: "Thermostable Alanine Racemase of Bacillus stearothermophilus" J.Biol.Chem.266-21. 13634-13639 (1991)

[Publications] 山田 秀明: "微生物の機能開発" 学会出版センタ-, 326 (1992)

小倉協三東北大学, 反応化学研究所, 教授 (80006303)

[Publications] 古山種俊: "イソプレノイド鎖延長酵素の機能" 日本農芸化学会誌. 65. 1791-1793 (1991)

[Publications] 山田秀明: "微生物の機能開発" 学会出版センタ-, 326 (1992)