Structure and Mechanism of LpxC in Lipid A Biosynthesis

描述（由申请人提供）：脂质A是脂多糖（LPS）的疏水性锚，是一种基于葡糖胺的磷脂，构成大多数革兰氏阴性菌外膜的外单层。也称为内毒素，脂质A是LPS的活性成分，其刺激免疫系统并引起危及生命的革兰氏阴性脓毒性休克，这是一种以弥散性血管内凝血和多器官衰竭为特征的严重病症。脂质A的生物合成是一个基本的途径，是保守的，几乎所有的革兰氏阴性菌。脂质A生物合成的关键步骤由UDP-3-O-（酰基）-N-乙酰葡糖胺脱乙酰酶（LpxC）催化。LpxC属于一个新的锌依赖性金属酰胺酶家族，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好靶标。事实上，LpxC的抑制导致细菌快速死亡，并治愈感染致死腹膜内剂量的大肠杆菌的小鼠（E. coh）。然而，来自E.大肠杆菌对来自其它革兰氏阴性菌的趋异LpxC相对无活性，特别是来自风产液菌和铜绿假单胞菌的趋异LpxC。虽然LpxCs已被广泛的生物化学研究和药理学筛选的主题，不寻常的抑制剂特异性和LpxCs及其复合物的结构信息的缺乏，无论是与底物或抑制剂，阻碍了进一步的机制研究LpxCs和优化其抑制剂。该提案的总体目标是揭示在脂质A生物合成中LpxC催化的基本未知的分子机制，并提供结构基础以合理化来自不同革兰氏阴性物种的LpxC的特异性。这些研究还将促进靶向LpxC的新型抗生素的开发。本研究的具体目标是：1）确定风产液菌LpxC（AaLpxC）的溶液结构; 2）确定AaLpxC/TU-514抑制剂复合物的溶液结构，并表征AaLpxC与底物的相互作用; 3）确定E. coil（EcLpxC）; 4）使用结构和生物化学方法表征EcLpxC与各种抑制剂之间的相互作用。

DESCRIPTION (provided by applicant): 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 lifethreatening 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. coli are relatively inactive against divergent LpxCs from other Gramnegative 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.