Mechanism of A5P Isomerase

描述（由申请人提供）：该资助旨在收集关于d -阿拉伯糖5-磷酸（ASP）异构酶（API）调控的机制信息，API是负责从d - 5-磷酸核酮糖（Ru5P）合成ASP的酶，在Gram-(-)微生物中。这一信息应证实，API是脂多糖（lps -又称内毒素）生物合成中的关键酶，是该领域研究人员筛选API选择性抑制剂的目标，即新一代机制多样且未知耐药性的抗生素。这个项目的目标是建立1。yrbH、kpsF、c3406和gutQ基因表达的ASP异构酶的胞内功能，以及三个基因产物共有的半胱甘氨酸-合成酶结构域（CBS）的调控作用；2. RuSP生成ASP的机理；和3。原料药的底物特异性和活性位点氨基酸的作用。具体目标集中在各种技术来检测中间烯二醇的存在，并确定底物和产物相互转化的立体化学。将构建基因组敲除以了解每种API的细胞功能以及API基因组敲除的潜在致命性（靶标验证）。将利用缺少CBS结构域的API的截断衍生物来确定CBS结构域的功能，并筛选糖核苷酸库以寻找CBS结构域的潜在调节因子。基于晶体学数据和使用基于机制的不可逆抑制剂对活性位点进行修饰的位点导向诱变研究，将被用于深入了解酶功能对底物结合、单体界面相互作用和API机制的贡献。在这项资助中合成的小分子将用作机械探针。这些研究的最终目标是更好地了解LPS的作用，LPS是一种对革兰氏（Gram-）微生物的生存和毒力都至关重要的关键大分子

DESCRIPTION (provided by applicant): 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 of mechanistically 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