Mechanism of A5P Isomerase

这项资助旨在收集有关D-阿拉伯糖5-磷酸化的机制信息，并了解其调控机制。 磷酸（ASP）异构酶（API），负责从D-核酮糖5- 磷酸盐（Ru 5 P），在革兰氏（-）微生物中。此信息应提供API（密钥） 脂多糖（LPS-aka内毒素）生物合成中的酶，是通过 本领域的研究人员鉴定了API的选择性抑制剂-即新一代的机械抑制剂 多种抗生素，已知没有耐药性。该项目的目标是建立1。的 由基因yrbH、kpsF、c3406和gutQ表达的ASP异构酶的细胞内功能，以及 为了了解胱硫醚β-合酶结构域（CBS）的调节作用， 基因产物; 2. RuSP生成ASP的机理; 3.的底物专一性 活性位点氨基酸的作用。具体目标集中在不同的技术来检测 存在的中间engly和确定立体化学的相互转化， 基质和产品。将构建基因组敲除以了解每个基因的细胞功能。 API以及API基因敲除的潜在致死性（靶标验证）。截断导数 将利用缺乏CBS结构域的API的100%来确定CBS结构域功能和糖库 将筛选核苷酸以发现CBS结构域的潜在调节剂。定点诱变 研究，基于晶体学数据和活性位点修饰，使用基于机制的不可逆 抑制剂，将被利用来深入了解酶功能对底物结合的贡献， 单体界面相互作用，以及API的作用机理。在这个过程中要合成的小分子 格兰特将作为机械探针。这些研究的最终目标是更好地了解 LPS是革兰氏（-）微生物生存和毒力的关键大分子

This grant proposes to collect mechanistic information on and understand the regulation of D-arabinose 5- phosphate (ASP) isomerase (API), the enzyme responsible for the synthesis of ASP from D-ribulose 5- phosphate (Ru5P), in Gram-(-) microorganisms. This information should provide validation that API, a key enzyme in lipopolysaccharide (LPS-aka endotoxin) biosynthesis, is a target for screening efforts by investigators in the field to identify selective inhibitors of API - namely a new generation ofmechanistically diverse antibiotics for which no resistance is known. The goals of this project are to establish 1. the intracellular function of the ASP isomerases expressed by the genes yrbH, kpsF, c3406 and gutQ as well as to understand the regulatory role of the cystathionine beta -synthase domain (CBS) common to three of the gene products; 2. the mechanism for the formation of ASP from RuSP; and 3. the substrate specificity of the APIs and the role of active site amino acids. The specific aims focus on diverse techniques to detect the presence of an intermediate enediol and to determine the stereochemistry of the inter-conversion of the substrate and product. Genomic knockouts will be constructed to understand the cellular function of each API as well as the potential lethality of a genomic knockout of API (target validation). Truncated derivatives of API lacking the CBS domain will be utilized to ascertain CBS domain function and a library of sugar nucleotides will be screened to find potential regulators of the CBS domain. Site-directed mutagenesis studies, based on crystallographic data and active site modification using mechanism-based irreversible inhibitors, will be exploited to gain insight into the contribution of enzyme functionalities to substrate binding, monomer interface interactions, and to the mechanism of API. The small molecules to be synthesized in this grant will serve as mechanistic probes. The ultimate goal of these studies is to better understand the role of LPS, a critical macromolecule essential to both the survival and virulence of Gram-(-) microorganisms