Protein engineering studies of thermostable D-amino acid transaminase

耐热D-氨基酸转氨酶的蛋白质工程研究

• 批准号: 05044095
• 负责人: SODA Kenji
• 金额: $ 3.84万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05044095/
• 关键词: thermophilic bacteria; thermostable enzymes; D-amino acid transaminase; pyridoxal phosphate; X-ray crystal structure analysis; pyridoxal enzymes; screening; protein engineering

We found that D-amino acid aminotransferase (D-AAT) of Bacillus sp.YM-1 and branched-chain L-amino acid aminotransferase (BCAT) of E.coli catalyze the re-face hydrogen transfer. These enzymes show a significant sequence homology with each other, but does not with all other aminotransferases. The three-dimensional structure of D-AAT demonstrated that the topographical situation of the catalytic residue of D-AAT is opposite to that of AspAT.The result of the crystallography also showed that the overall fold of D-AAT is quite different from those of AspAT and other aminotransferases, whose structures resemble each other. We established a simple method for determination of the stereospecificity of C-4' hydrogen transfer of the coenzyme based on these findings. We developed also a general procedure to determine the common free D-amino acids except D-proline by means of D-AAT and 2-oxohexanoate : the formation of norleucine denotes the presence of some D-amino acid (s) whose identity can be established by a corresponding decrease in the susceptible amino acid (s) after treatment. We found that D-AAT is inactivated by incubation with D-aspartate, D-glutamate and D-alanine, the best substrates, and that the inactivation is accompanied by the slow release of the cx-carboxylg group of these amino acids. Lys-145, which binds pyridoxal-P,is not involved in the inactivation since K145Q and K145N mutanat enzymes are also inactivated. We studied the catalytic role of Leu-201, the residue in the vicinity of the active site to interact with the bound pyridoxal-P.The L201A and L201W mutant enzymes showed anomalous kinetic behavior. These show that Leu-201 regulates the function of cofactor during the reaction of D-AAT.

我们发现芽孢杆菌（Bacillus sp. m . 1）的d-氨基酸转氨酶（D-AAT）和大肠杆菌（E.coli）的支链l -氨基酸转氨酶（BCAT）可以催化氢的再表面转移。这些酶具有显著的序列同源性，但与所有其他转氨酶不具有同源性。D-AAT的三维结构表明，D-AAT催化残渣的地形情况与AspAT相反。结晶学结果还表明，D-AAT的整体折叠与AspAT和其他转氨酶有很大的不同，它们的结构相似。基于这些发现，我们建立了一种简单的方法来测定辅酶C-4'氢转移的立体特异性。我们还开发了一个通用的程序来确定除d -脯氨酸以外的常见游离d -氨基酸，通过D-AAT和2-氧己酸盐：去甲氨酸的形成表明存在一些d -氨基酸，其身份可以通过处理后敏感氨基酸的相应减少来确定。我们发现D-AAT与d -天冬氨酸、d -谷氨酸和d -丙氨酸这三种最佳底物孵育后失活，并且失活伴随着这些氨基酸的cx-羧基的缓慢释放。结合pyridoxa - p的Lys-145不参与失活，因为K145Q和K145N突变酶也失活。我们研究了活性位点附近的残基Leu-201与结合的吡哆醇- p相互作用的催化作用。L201A和L201W突变体酶表现出异常的动力学行为。说明在D-AAT的反应过程中，Leu-201调节了辅因子的功能。

项目成果

Wanda M.Jones et al.: "Determination of Free D-Amino Acids with a Bacterial Transaminase ; Their Depletion Leads to Inhibition of Bacterial Growth" Anal.Biochem.218. 204-209 (1994)

Wanda M.Jones 等人：“用细菌转氨酶测定游离 D-氨基酸；它们的消耗导致细菌生长的抑制”Anal.Biochem.218。

K.Kishimoto et al.: "Role of Leucine 201 of Thermostable D-Amino Acid Aminotransferase from a Thermophile, Bacillus sp.YM-1" J.Biochem.(in press). (1994)

K.Kishimoto 等人：“来自嗜热芽孢杆菌属 sp.YM-1 的热稳定 D-氨基酸氨基转移酶的亮氨酸 201 的作用”J.Biochem.（出版中）。

K.Soda et al.: "Biochemistry of Vitamin B6 and PQQ" Birkhauser Verlag Basel/Switzerland, (1994)

K.Soda 等人：“维生素 B6 和 PQQ 的生物化学”Birkhauser Verlag 巴塞尔/瑞士，(1994)

Dong-Woon Kim: "Studies of the Active-Site Lrsrl Residue of Thermostable Aspartate Aminotransferase:Combination of Site-Directed Mutagenesis and Chemical Modification" The Journal of Biochemistry. 115. 93-97 (1994)

Dong-Woon Kim：“热稳定天冬氨酸转氨酶活性位点 Lrsrl 残基的研究：定点诱变和化学修饰的结合”《生物化学杂志》。

K.Soda et al.: "Pyridoxal Enzymes Acting on D-Amino Acids" Pure & Appl.Chem.66. 709-714 (1994)

K.Soda 等人：“作用于 D-氨基酸的吡哆醛酶”纯