GENOMIC ENZYMOLOGY--THE ENOLASE SUPERFAMILY

基因组酶学——烯醇酶超家族

• 批准号: 6525891
• 负责人: JOHN A GERLT
• 金额: $ 42.46万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-02 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525891
• 关键词: X ray crystallography; active sites; catalyst; enzyme activity; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; glutarates; phosphopyruvate hydratase; stereochemistry

DESCRIPTION: The members of the enolase superfamily catalyze different overall reactions that share a common partial reaction in which a stabilized enolate anion is generated by abstraction of the alpha-proton of a carboxylic acid. The enolate anion intermediates are partitioned to different products in the different active sites. The catalytic groups in the structurally characterized members of the superfamily are found in (beta/alpha)-barrel domains. The principal investigator and his group describe structural and mechanistic studies of new members of the superfamily that will provide an understanding of the general strategy used to catalyze the common partial reaction and the subsequent divergent partial reactions to generate different products: 1) D-Glucarate dehydratase from Escherichia coli (GlucD) will be studied so that they can understand how the structure of the barrel domain can be modified to evolve an active site that is homologous to mandelate racemate but additionally catalyzes a dehydration reaction. 2) D-Galactonate dehydratase (GalD) and L-rhamnonate dehydratase (RhamD), both from E. coli, catalyze dehydration reactions on acid sugar substrates that differ in the stereochemistry of the leaving OH group. Dr. Gerlt and his group will study these so that they can determine how Nature varied the structure of the barrel domain to allow the stereochemical courses of this dehydration reaction to differ. 3) Orthologous o-succinylbenzoate synthases (OSBS's) are related by a low degree of sequence identity. The OSBS from Amycolaptosis also catalyzes the racemization of N-acylamino acids. They will explore the hypothesis that the sequence divergence is possible because the substrate is chemically unstable. They will improve the racemase activity of the OSBS from Amycolaptosis, thereby mimicking the process that Nature used to evolve new enzymes from old enzymes. 4) Although carboxyphosphonoenolpyruvate synthase from Streptomyces hygroscopicus (CPEPS) is homologous to enolase, its primary sequence lacks the essential general acid catalyst in the enolase. CPEPS is reported to catalyze a transesterification reaction. Dr. Gerlt and his group will investigate the mechanism of this reaction so that they can better understand the underlying catalytic strategy used by the members of the superfamily.

描述：烯醇化酶超家族的成员催化不同的整体 共享共同部分反应的反应，其中稳定的烯醇化物 阴离子通过夺取羧酸的α-质子而产生。的 烯醇化阴离子中间体在反应器中被分配成不同的产物， 不同的活性部位。结构表征的催化剂中的催化基团 该超家族的成员存在于（β/α）-桶结构域中。的 首席研究员和他的团队描述了结构和机械 对超家族新成员的研究， 用于催化常见部分反应的一般策略和 随后的发散部分反应以产生不同的产物：1） 将研究来自大肠杆菌的D-葡糖二酸脱氢酶（GlucD）， 他们可以理解桶域的结构如何被修改， 进化出一个活性位点，与扁桃酸根同源，但另外 催化脱水反应。2)D-半乳糖酸脱氢酶（GalD）和 L-鼠李糖酸脱氢酶（RhamD）均来自E.大肠杆菌，催化脱水 在立体化学上不同的酸性糖底物上的反应 留下OH基团。Gerlt博士和他的团队将研究这些， 确定大自然如何改变桶域的结构， 这种脱水反应的立体化学过程不同。3)直系同源 邻-琥珀酰苯甲酸酯酶（OSBS's）通过低程度的序列相关 身份来自Amycolaptosis的OSBS还催化 N-酰基氨基酸。他们将探索一种假设， 发散是可能的，因为衬底是化学不稳定的。他们将 提高来自无菌凋亡的OSBS的消旋酶活性，从而模拟 大自然用来从旧酶进化出新酶的过程。四、 虽然来自吸水链霉菌的羧基膦酰基烯醇丙酮酸合酶 （CPEPS）与烯醇化酶同源，其一级序列缺乏必需的氨基酸序列。 烯醇化酶中的一般酸性催化剂。据报道，CPEPS催化 酯交换反应Gerlt博士和他的团队将调查 这种反应的机制，以便他们能够更好地了解潜在的 超家族成员使用的催化策略。