Structure and Mechanism of LpxC in Lipid A Biosynthesis

描述（由申请人提供）：脂质A，脂多糖（LPS）的疏水锚点，是一种以氨基葡萄糖为基础的磷脂，构成大多数革兰氏阴性菌外膜的外单层。脂质A也被称为内毒素，是LPS的活性成分，刺激免疫系统并导致危及生命的革兰氏阴性感染性休克，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疾病。脂质A生物合成是几乎所有革兰氏阴性菌中保守的重要途径。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。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一个很好的靶点。事实上，抑制LpxC会导致细菌快速死亡，并治愈腹腔内致命剂量的埃希氏杆菌（E. coh）感染的小鼠。然而，对来自大肠杆菌的LpxC的有效抑制剂对来自其他革兰氏阴性菌的发散性LpxC相对无效，特别是来自风水蛭和铜绿假单胞菌的LpxC。尽管LpxCs已经成为广泛的生化研究和药理学筛选的主题，但LpxCs及其复合物（无论是与底物还是抑制剂）不寻常的抑制剂特异性和结构信息的缺乏，阻碍了对LpxCs的进一步机制研究和抑制剂的优化。本研究的总体目标是揭示LpxC在脂质A生物合成中催化作用的未知分子机制，并为理顺不同革兰氏阴性物种LpxCs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。具体目标是：1)确定Aquifex aeolicus （AaLpxC）中LpxC的溶液结构；2)确定AaLpxC/TU-514抑制剂配合物的溶液结构，表征AaLpxC与其底物的相互作用；3)测定e线圈中LpxC的溶液结构（EcLpxC）；4)利用结构和生化方法表征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.