Protein Engineering of Aspartate Aminotransferase

天冬氨酸转氨酶的蛋白质工程

• 批准号: 03670132
• 负责人: KURAMITSU Seiki
• 金额: $ 1.28万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670132/
• 关键词: Enzyme; Catalytic Mechanism; Substrate Recogition; Protein Engineering; Aminotransferase; Chimera; X-Ray Crystallography; Site-Directed Mutagenesis; 量産化; アスパラギン酸アミノトランスフェラ-ゼ

Aminotransferases catalyze transamination between amino acids and alphaketo acids. Escherichia coli aspartate aminotransferase (AspAT) has high specificity for acidic substrates, whereas E. coli aromatic amino acid aminotransferase (AroAT) has high specificity for both acidic and hydrophobic substrates. The two proteins have only 40% amino acid sequence homology, and 50% nucleotide sequence homology. X-ray crystallographic studies of E. coli AspAT have revealed the binding site for the acidic substrate, but that for the hydrophobic substrate is still unknown. Site-directed mutagenesis of AspAT or AroAT has failed to reveal the hydrophobic pocket. Therefore, in order to search for the hydrophobic pocket, we constructed AspAT-AroAT chimeric proteins.The chimeric proteins were obtained, even though the homology of the nucleotides was as low as 50%. The yield of chimeric gene was related to the length of homologous region between the two aminotransferase.The recombination occurred in the region where eight nucleotides out of ten were identical. It is suggested that the amino acid residues distant from active site contribute also contribute to the substrate specificity of aminotransferase.

氨基转移酶催化氨基酸和α-酮酸之间的转氨作用。大肠杆菌天冬氨酸氨基转移酶（AspAT）对酸性底物具有高度特异性，而大肠杆菌AspAT对酸性底物具有高度特异性。大肠杆菌芳香族氨基酸氨基转移酶（AroAT）对酸性和疏水性底物都具有高度特异性。这两种蛋白的氨基酸序列同源性只有40%，核苷酸序列同源性只有50%。X射线晶体学研究表明，E. coliAspAT已经揭示了酸性底物的结合位点，但疏水底物的结合位点仍然未知。AspAT或AroAT的定点诱变未能揭示疏水口袋。因此，为了寻找疏水口袋，我们构建了AspAT-AroAT嵌合蛋白，获得了嵌合蛋白，尽管核苷酸同源性低至50%。嵌合基因的产量与两个氨基转移酶同源区的长度有关，重组发生在10个核苷酸中有8个核苷酸相同的区域。这表明，远离活性位点的氨基酸残基也有助于氨基转移酶的底物特异性。

项目成果

倉光 成紀: "新生化学実験講座 第1巻 VII.タンパク質工学" 東京化学同人, 33 (1993)

仓光茂德：《新化学实验教程第1卷VII.蛋白质工程》东京化学同人，33（1993）

倉光 成紀: "蛋白質・核酸・酵素37(No.13)p2243-2256" 共立出版, 14 (1992)

Shigenori Kuramitsu：“蛋白质、核酸和酶 37（No.13）p2243-2256”Kyoritsu Shuppan，14（1992）

倉光 成紀(共著): "新生化学実験講座第1巻VII.タンパク質工学" 東京化学同人, 33 (1993)

Shigenori Kuramitsu（合着）：《新化学实验课程第1卷VII.蛋白质工程》东京化学同人，33（1993）

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Sung, M.-H.：“来自嗜热芽孢杆菌属物种的耐热天冬氨酸转氨酶的分子克隆和序列测定”“依赖于磷酸吡哆醛和其他羰基化合物作为辅因子的酶”（Fukui, T.、Kagamiyama, H.、Soda , K .,&

OKAMOTO,A.: "Three-Dimensional Structure of Aspartate Aminotransferase from Escherichia coli" ″Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors″(Fukui,T.,Kagamiyama,H.,Soda,K.,& Wada,H.,eds.). 107-109 (1991)

OKAMOTO, A.：“大肠杆菌天冬氨酸转氨酶的三维结构”“依赖于磷酸吡哆醛和其他羰基化合物作为辅因子的酶”（Fukui, T.、Kagamiyama, H.、Soda, K. 和 Wada） ，H.，编辑）107-109（1991）。