Mechanism of A5P Isomerase

描述(由申请人提供)：这项资助建议收集关于D-阿拉伯糖5-磷酸(ASP)异构酶(API)的机制信息并了解其调节，API是负责从D-核酮糖(Ru5P)合成D-阿拉伯糖5-磷酸(Ru5P)的酶，在革兰氏(-)微生物中。这些信息应该证实，API是内毒素(LPS)生物合成的关键酶，是该领域研究人员努力筛选的目标，以确定API的选择性抑制剂-即机制多样化的新一代抗生素，目前尚不清楚其耐药性。本项目的目标是：1.建立由yrbH、KPSF、C3406和gutQ基因表达的天冬氨酸异构酶的细胞内功能，以及了解其中三个基因产物所共有的胱硫醚-β-合成酶结构域(CBS)的调节作用；2.从RuSP形成天冬氨酸异构酶的机制；3.APIs的底物特异性和活性部位氨基酸的作用。具体目标侧重于各种技术，以检测中间体烯二醇的存在，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)。缺少CBS结构域的API的截断衍生物将被用来确定CBS结构域的功能，并将筛选糖核苷酸文库以寻找CBS结构域的潜在调控因子。定点突变研究，基于结晶学数据和使用基于机理的不可逆抑制剂的活性位修饰，将被用来深入了解酶功能对底物结合、单体界面相互作用的贡献，以及对API的机理的影响。将在这项拨款中合成的小分子将用作机械探测器。这些研究的最终目标是更好地了解内毒素的作用，内毒素是一种对革兰氏(-)微生物的生存和毒力至关重要的关键大分子

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