Mechanism of A5P Isomerase

这项拨款建议收集关于D-阿拉伯糖5-的机制信息并了解其调节。 磷酸(ASP)异构酶(API)，负责从D-核酮糖合成天冬氨酸异构酶。 革兰氏(-)微生物中的磷酸盐(Ru5P)。此信息应提供验证API、密钥 内毒素生物合成中的酶，是筛选努力的目标，通过 该领域的研究人员正在寻找API的选择性抑制剂--即机械上的新一代 抗生素种类繁多，目前尚不清楚其抗药性。这个项目的目标是建立1. YrbH、kpsf、c3406和gutQ基因表达的天冬氨酸异构酶及其胞内功能 为了了解胱硫醚β-合成酶结构域(CBS)的调节作用， 基因产物；2.RuSP形成ASP的机制；3.RuSP的底物特异性 APIs及其活性部位氨基酸的作用。具体目标集中在各种技术上，以检测 中间体烯二醇的存在，并确定相互转化的立体化学 底物和产品。将构建基因组敲除以了解每个基因的细胞功能 以及API基因组敲除的潜在致命性(靶标验证)。截断导数 将利用缺少CBS结构域的API来确定CBS结构域的功能和糖库 将对核苷酸进行筛选，以寻找CBS结构域的潜在调控因子。定点突变 基于结晶学数据和基于机理的不可逆活性中心修饰的研究 抑制剂，将被用来洞察酶官能团对底物结合的贡献， 单体间的界面相互作用，以及API的作用机理。在这种情况下合成的小分子 格兰特将充当机械探测器。这些研究的最终目标是更好地理解 内毒素，一种对革兰氏(-)微生物的生存和毒力至关重要的关键大分子

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