Origine and Molecular Evolutionary Engineering of the Enzyme-Coenzyme Systems

酶-辅酶系统的起源和分子进化工程

• 批准号: 08680683
• 负责人: YOSHIMURA Tohru
• 金额: $ 1.54万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680683/
• 关键词: coenzyme; molecular evolution; pyridoxal phosphate; stereochemistry; aminotransafearse; lyase; glutamate racemase

The stereospecificities for the hydrogen transfer between C-4' of the cofactor and substrate in the transamination catalyzed by various pyridoxal 5'-phosphate (PLP)-dependent enzymes have been studied. The stereospecificities reflect the active-site structures of the enzymes, especially the topographical situation of a coenzyme-substrate Schiff base and a catalytic base for the hydrogen transfer. We found that three types of enzymes catalyzing the reaction stereospecifically on either si-or re-face of the planar intermediate, and alternatively non-stereospecifically on both faces. The classification of PLP-enzymes based on the stereospecificcity for the hydrogen transfer coincident with that based on their primary and three dimensional structures. These suggest that PLP-enzymes have been evolved from at least three different ancestral proteins. These studies lead us the speculation that enzymes requiring coenzymes were created by the combination of the inherently active coenzymes and protein offering the place of the reactions. We attempted to produce a new enzyme by combining a hemin and glutamate recemase showing a significant similarity with mammalian myoglobins in their primary structures.

研究了各种吡哆醛 5'-磷酸 (PLP) 依赖性酶催化的转氨基作用中辅因子 C-4' 和底物之间氢转移的立体特异性。立体特异性反映了酶的活性位点结构，特别是辅酶底物希夫碱和氢转移催化碱的拓扑情况。我们发现三种类型的酶在平面中间体的硅面或再面上立体定向地催化反应，或者在两个面上非立体定向地催化反应。基于氢转移立体特异性的 PLP 酶分类与基于其一级和三维结构的分类一致。这些表明 PLP 酶是从至少三种不同的祖先蛋白质进化而来的。这些研究使我们推测，需要辅酶的酶是通过固有活性辅酶和提供反应场所的蛋白质的组合而产生的。我们试图通过结合血红素和谷氨酸还原酶来生产一种新的酶，其初级结构与哺乳动物肌红蛋白显着相似。

项目成果

Lim, Y.-H.: "Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity" The Journal of Biological Chemistry. 273巻7号. 4011-4015 (1998)

Lim, Y.-H.：“广泛底物特异性的磷酸吡哆醛依赖性氨基酸消旋酶催化的非立体特异性转氨”《生物化学杂志》，第 273 卷，第 7 期。4011-4015 (1998)

Jhee, K.H.et al.: "Stereospecificity of Thermostable Ornithine 5-Aminotransferase for the Hydrogen Transfer in the L-and D-Ornithine Transamination" Biochemistry. 35(30). 9792-9796 (1996)

Jhee, K.H. 等人：“热稳定鸟氨酸 5-氨基转移酶在 L-和 D-鸟氨酸转氨作用中氢转移的立体特异性”生物化学。

Lim,Y.-H.: "Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity" The Journal of Biological Chemistry. 273巻7号. 4011-4015 (1998)

Lim, Y.-H.：“广泛底物特异性的磷酸吡哆醛依赖性氨基酸消旋酶催化的非立体特异性转氨”《生物化学杂志》，第 273 卷，第 7 期。4011-4015 (1998)

Lim, Y.H.: "Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity" J.Biol.Chem.273(7). 4011-4015 (1998)

Lim, Y.H.：“具有广泛底物特异性的磷酸吡哆醛依赖性氨基酸消旋酶催化的非立体特异性转氨作用”J.Biol.Chem.273(7)。

Liu,L.: "Cloning and Expression of the Glutamate Racemase Gene of Bacillus pumilus" Journal of Biochemistry. 121巻6号. 1155-1161 (1997)

Liu, L.：“短小芽孢杆菌谷氨酸消旋酶基因的克隆和表达”，生物化学杂志，第 121 卷，第 6 期，1155-1161 (1997)。