Structural studies on enzymes involved in the formation of salicylate and p-aminobenzoate

水杨酸和对氨基苯甲酸形成酶的结构研究

• 批准号: BB/D011701/1
• 负责人: Chris Abell
• 金额: $ 28.81万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD011701%2F1
• 关键词: Structural; studies; enzymes; involved; formation

A number of different aromatic molecules are made from chorismate. We are interested in the enzymes that use this molecule and convert it into different products. What do the enzymes look like (what is their 3D protein crystal structure)? How does one enzyme differ from another? Can we interpret these differences to explain the different reactions they catalyse? We plan to study two systems: (a) the enzymes that convert chorismate to salicylate, and (b) the enzymes that convert chorismate to ADC, an intermediate on the way to p-aminobenzoate. The conversion of chorismate to salicylate involves two steps. The conversion of chorismate to isochorismate, and the conversion of isochorismate to salicylate. We have recently solved the first structure of a 'salicylate synthase', an enzyme (Irp9) that does both of these steps. We now plan to solve the crystal structures of an enzyme that just catalyses the first step (called EntC), and one that just catalyses the second step (PchB). We also plan to make variants of Irp9 to get a better understanding of how it works. The conversion of chorismate to ADC also involves two steps. The first is catalysed by PabA. It converts glutamine to ammonia and glutamate. The ammonia is then thought to pass through a tunnel to get to the active site of PabB, where it reacts with chorismate to make ADC. We plan to solve the crystal structure of the PabA:PabB complex to see if we can get information about this tunnel. We also hope to get a crystal structure of a novel reaction intermediate attached to PabB that we have previously detected by mass spectrometry. We will learn a great deal from these two projects. We will have a better understanding of why some enzymes catalyse one reaction and then stop, while another apparently similar enzyme, catalyses two reactions. We will also learn something about how the reactions proceed by getting the structure of intermediates and analogues bound at the active sites of the enzymes

许多不同的芳香分子是由氯酸盐组成的。我们感兴趣的是利用这种分子并将其转化为不同产物的酶。酶是什么样子的（它们的三维蛋白质晶体结构是什么）？一种酶与另一种酶有何不同？我们能解释这些差异来解释它们催化的不同反应吗？我们计划研究两个体系：(a)将choris酸转化为水杨酸的酶，(b)将choris酸转化为ADC的酶，ADC是合成对氨基苯甲酸酯的中间体。把氯酸盐转化为水杨酸盐需要两个步骤。氯酸盐转化为异氯酸盐和异氯酸盐转化为水杨酸盐。我们最近解决了“水杨酸合酶”的第一个结构，这是一种完成这两个步骤的酶（Irp9）。我们现在计划解决一种酶的晶体结构，这种酶只催化第一步（称为EntC），另一种酶只催化第二步（PchB）。我们还计划制作Irp9的变体，以便更好地了解它是如何工作的。chorismate到ADC的转换也包括两个步骤。第一种是由PabA催化的。它将谷氨酰胺转化为氨和谷氨酸。然后，氨被认为通过一个通道到达pab的活性位点，在那里它与choris酸盐反应生成ADC。我们计划解决PabA: pab复合物的晶体结构，看看我们是否能得到关于这个隧道的信息。我们也希望得到一个新的反应中间体的晶体结构，连接到pab，我们已经通过质谱检测到。我们将从这两个项目中学到很多东西。我们将更好地理解为什么有些酶催化一个反应然后停止，而另一个表面上相似的酶催化两个反应。我们还将通过了解酶活性位点的中间产物和类似物的结构来了解反应是如何进行的

项目成果

Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase (EntC) bound to its reaction product isochorismate: implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes.

• DOI: 10.1016/j.jmb.2010.01.019
• 发表时间: 2010-03
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: S. Sridharan;N. Howard;O. Kerbarh;M. Błaszczyk;C. Abell;T. Blundell