Mechanistic Studies of a Bacterial Capsule Polymerase

项目总结 本提案旨在研究奈瑟氏奈瑟菌胶囊聚合酶的催化作用。 脑膜炎，脑膜炎的主要细菌原因之一。这个包裹着细菌的胶囊 富含多糖。血清组特有的酶负责合成这些 碳水化合物。合成单糖聚合物的胶囊酶一直是 与那些合成含有两种不同糖的聚合物相比，研究得很好。作为一名 结果，对这些杂多聚合体胶囊聚合酶的催化作用知之甚少。 酵素。这类酶为疫苗研究和药物提供了一个靶点。 发展。这项工作的总体目标是加深对奈瑟里亚的了解 脑膜炎W群被膜多聚酶，形成多糖 含有两种不同糖的交替重复的。我们这项工作的目标是执行一系列 将为这种酶的机制提供深入了解的基础实验。这一目标将 通过以下具体目标来实现： 目的1：测定脑膜炎奈瑟菌W群胶囊的催化动力学 聚合酶。我们将使用基于高效液相的荧光分析来进行动力学测量。 为了进行这项检测，我们首先要确定最佳的荧光受体。《知识》 通过完成这一目标(酶的动力学参数[Km，Vmax，Kcat])获得 目前尚不清楚)，将提供对催化速率和机理的重要洞察。 目的2：测定脑膜炎奈瑟氏菌W群胶囊的关键氨基酸 聚合酶催化。我们将用化学酶法合成一种可光激活的底物 模拟，CMP-SiaDAz，将用于实现结合位点的光活化标记 残留物。我们将通过质谱学确定修饰氨基酸的位置 分析并进行蛋白质突变形式的活性分析。所获得的知识将 为酶催化过程中的底物识别和结合提供了新的见解。 目的三：利用末端核苷酸供体合理控制 多糖类合成。我们将使修饰的核苷酸供体糖与酶反应。 并评估不同条件下的多糖组成/链长。完成 这个目标将揭示这种酶如何与替代底物反应，从而产生新的潜力。 酶抑制剂。

PROJECT SUMMARY This proposal seeks to investigate catalysis of a capsule polymerase enzyme from Neisseria meningitidis, one of the leading bacterial causes of meningitis. This capsule around the bacteria is rich in polysaccharides. Serogroup-specific enzymes are responsible for synthesis of these carbohydrates. The capsule enzymes that synthesize polymers of a single sugar have been well studied compared to those that synthesize polymers containing two different sugars. As a result, much less is known about catalysis by these heteropolymeric capsule polymerase enzymes. This class of enzymes provides one target for vaccine research and drug development. The overall goal of this work is to develop an understanding of the Neisseria meningitidis serogroup W capsule polymerase enzyme which makes a polysaccharide containing alternate repeats of two different sugars. Our goal in this work is to perform series of fundamental experiments that will provide insight into this enzyme's mechanism. This goal will be accomplished by the following specific aims: Aim 1: Determine the kinetics of catalysis by the N. meningitidis serogroup W capsule polymerase. We will perform kinetics measurements using an HPLC-based fluorescent assay. To carry out this assay, we will first define optimal fluorescent acceptors. The knowledge gained from completion of this aim (kinetic parameters [Km, Vmax, kcat] of the enzyme that are currently unknown), will provide important insight into catalytic rates and mechanism. Aim 2: Determine key amino acids critical to N. meningitidis serogroup W capsule polymerase catalysis. We will chemoenzymatically synthesize a photoactivatable substrate analog, CMP-SiaDAz, which will be used to achieve photoactivated labeling of binding site residues. We will determine the location of modified amino acids by mass spectrometry analysis and perform activity assay of mutant forms of the protein. The knowledge gained will provide new insight into substrate recognition and binding during enzyme catalysis. Aim 3: Use chain-terminating nucleotide sugar donors to rationally control polysaccharide synthesis. We will react modified nucleotide donor sugars with the enzyme under various conditions and assess polysaccharide composition/chain length. Completion of this aim will reveal how this enzyme can react with alternate substrates leading to new potential enzyme inhibitors.