Structure and Mechanism of LpxC in Lipid A Biosynthesis

超出提供的空间。脂类A是脂多糖的疏水锚，是一种以氨基葡萄糖为基础的磷脂，构成了大多数革兰氏阴性菌外膜的外单分子层。也被称为内毒素，脂类A是内毒素的活性成分，刺激免疫系统，导致危及生命的革兰氏阴性败血症休克，这是一种以弥漫性血管内凝血和多器官衰竭为特征的严重疾病。脂质A的生物合成是一种基本的途径，几乎在所有革兰氏阴性生物中都是保守的。脂质A生物合成的关键步骤是由UDP-3-O-(酰基)-N-乙酰氨基葡萄糖脱乙酰酶(LpxC)催化的。LpxC属于一个新的锌依赖金属酰胺酶家族，与任何已知的哺乳动物蛋白没有序列同源性。因此，它是设计新型抗生素的极佳靶点。事实上，抑制LpxC会导致细菌迅速死亡，并治愈被致命剂量的大肠杆菌(E.coh)感染的小鼠。然而，来自E.coil的LpxC的有效抑制剂对来自其他革兰氏阴性菌的LpxC相对无效，特别是来自Aquifex aeolicus和铜绿假单胞菌的LpxC。尽管LpxCs已经成为广泛的生化研究和药理筛选的对象，但LpxCs及其与底物或抑制剂的复合体的不寻常的抑制剂专一性和结构信息的缺乏阻碍了对LpxCs的进一步机制研究和其抑制剂的优化。这项建议的总体目标是揭示LpxC在脂质A生物合成中催化作用的分子机制，并为合理确定不同革兰氏阴性菌LpxC的特异性提供结构基础。这些研究也将促进针对LpxC的新型抗生素的开发。在拟议的工作中，具体目标是：1)确定Aquifex aeolicus LpxC(AaLpxC)的溶液结构；2)确定AaLpxC/TU-514抑制剂复合体的溶液结构并表征AaLpxC与其底物之间的相互作用；3)确定E.Coil LpxC(EcLpxC)的溶液结构；4)用结构和生化方法表征EcLpxC与各种抑制剂的相互作用。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is a glucosamine-based phospholipid that constitutes the outer monolayer of the outer membrane of most Gram-negative bacteria. Also known as endotoxin, lipid A is the active component of LPS that stimulates the immune system and causes life- threatening Gram-negative septic shock, a severe condition characterized by disseminated intra-vascular coagulation and multiple organ failure. Lipid A biosynthesis is an essential pathway that is conserved in virtually all Gram-negative organisms. The committed step of lipid A biosynthesis is catalyzed by UDP-3-O- (acyl)-N-acetylglucosamine deacetylase (LpxC). LpxC belongs to a novel family of zinc-dependent metalloamidases and shares no sequence homology with any known mammalian proteins. Hence, it is an excellent target for the design of novel antibiotics. Indeed, inhibition of LpxC causes rapid bacterial death and cures mice infected with a lethal intraperitoneal dose of Escherichia coil (E. coh). However, potent inhibitors against the LpxC from E. coilare relatively inactive against divergent LpxCs from other Gram- negative bacteria, particularly, those from Aquifex aeolicus and Pseudomonas aeruginosa. Although LpxCs have been the subject of extensive biochemical studies and pharmacological screenings, the unusual inhibitor specificity and the lack of structural information on LpxCs and their complexes, either with substrates or inhibitors, hinder further mechanistic studies on LpxCs and the optimization of their inhibitors. The overall goal of this proposal is to reveal the largely unknown molecular mechanism underlying LpxC catalysis in lipid A biosynthesis and to provide a structural basis to rationalize the specificity of LpxCs from different Gram-negative species. These studies should also facilitate the development of novel antibiotics targeting LpxC. In the proposed work, the specific aims are: 1) determining the solution structure of the LpxC from Aquifex aeolicus (AaLpxC); 2) determining the solution structure of the AaLpxC/TU-514 inhibitor complex and characterizing the interaction between AaLpxC and its substrate; 3) determining the solution structure of the LpxC from E. coil (EcLpxC); 4) characterizing the interactions between EcLpxC and various inhibitors using structural and biochemical approaches. PERFORMANCE SITE ========================================Section End===========================================