Mechanistic Studies of a Bacterial Capsule Polymerase

细菌荚膜聚合酶的机理研究

• 批准号: 9751322
• 负责人: Pumtiwitt McCarthy
• 金额: $ 15.1万
• 依托单位: MORGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-26 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751322
• 关键词: Amino Acids; Area; Bacteria; Bacterial Capsules; Bacterial Meningitis; Binding; Binding Sites; Biological Assay; Carbohydrates; Catalysis; Development; Disease; Enzymatic Biochemistry; Enzyme Inhibitor Drugs; Enzymes; Family; Foundations; Future; Glycoconjugates; Goals; Gram-Negative Bacteria; High Pressure Liquid Chromatography; Kinetics; Knowledge; Label; Length; Location; Mass Spectrum Analysis; Measurement; Medical; Meningitis; Molecular; Molecular Probes; Neisseria; Neisseria meningitidis; Nucleotides; Organism; Pathogenicity; Polymerase; Polymers; Polysaccharides; Property; Proteins; Recombinants; Route; Series; Structure; Surface; Therapeutic Intervention; Vaccine Research; Vaccines; Work; analog; base; capsule; carbohydrate biosynthesis; design; drug development; enzyme activity; enzyme mechanism; experimental study; glycosyltransferase; insight; member; mutant; research and development; sugar; sugar nucleotide; targeted treatment; tool

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.

项目摘要 该建议旨在研究奈瑟氏菌的荚膜聚合酶的催化作用 脑膜炎，脑膜炎的主要细菌原因之一。这个围绕着细菌的胶囊 富含多糖。血清群特异性酶负责合成这些 碳水化合物合成单一糖聚合物的胶囊酶已经被发现 与那些合成含有两种不同糖的聚合物的方法相比，研究得很好。作为 结果，对这些多聚体胶囊聚合酶的催化作用知之甚少 内切酶这类酶为疫苗研究和药物开发提供了一个靶点。 发展这项工作的总体目标是发展对奈瑟菌的了解， 脑膜炎血清群W荚膜聚合酶，其产生多糖 含有两种不同糖的交替重复。我们在这项工作中的目标是执行一系列 基础实验将提供对这种酶的机制的深入了解。这一目标将 通过以下具体目标实现： 目的1：测定N. W群脑膜炎病毒胶囊 聚合酶。我们将使用基于HPLC的荧光测定法进行动力学测量。 为了进行这种测定，我们将首先确定最佳的荧光受体。知识 从完成这一目标获得的（动力学参数[Km，Vmax，kcat]的酶， 目前未知），将提供重要的洞察催化速率和机制。 目的2：确定N. W群脑膜炎病毒胶囊 聚合酶催化我们将用化学酶法合成一种光活化底物 类似物，CMP-SiaDAz，其将用于实现结合位点的光活化标记 残基我们将通过质谱法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测定。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3：利用链终止核苷酸糖供体合理控制 多糖合成我们将使修饰的核苷酸供体糖与酶反应 并评估多糖组成/链长。完成 这一目标将揭示这种酶如何与其他底物反应，从而产生新的潜力。 酶抑制剂