BIOSYNTHESIS OF MEMBRANE GLYCOLIPIDS IN RHIZOBIUM

根瘤菌膜糖脂的生物合成

基本信息

批准号：
6519607
负责人：
Christian R Raetz
金额：
$ 29.67万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2004-06-30
项目状态：
已结题

项目摘要

Lipopolysaccharides (LPSs) are remarkable glycolipids that comprise the outer surfaces of Gram-negative bacteria, including the symbiotic organism, Rhizobium leguminosarum. In Escherichia coli, the lipid A anchor of LPS is a hexa-acylated disaccharide of glucosamine, bearing phosphate moieties at positions 1 and 4'. The minimal LPS required for a growth of E.coli consists of lipid A and two extra sugars. Emerging genomic sequences indicate that the enzymes that make lipid A in E. coli are present in most other Gram-negative bacteria. Lipid A (often termed endotoxin) is also the active component of LPS responsible for the clinical complications of Gram-negative sepsis. Minor modifications in the structure of lipid A can have profound effects on pathogenesis. Some lipid A analogs are actually potent endotoxin antagonists. Compared to E. coli, the chemical structures of the lipid A and core domains of R. leguminosarum LPS are very unusual. R. leguminosarum lipid A lacks the and 4'phosphates, but is modified with galacturonic acid at position 4'. It is acylated with a peculiar 28 carbon fatty acid, and contains 2-deoxy-2-aminogluconate in place of the proximal glucosamine. The structure of R. leguminosarum LPS indicates the existence of novel enzymes for generating diverse lipid A and core species. It is now established that the first seven enzymes of lipid A biosynthesis are in fact the same in E. coli and R. leguminosarum. The differences arise in the later stages of the pathway. To date, enzymes identified as unique to R. leguminosarum include a 4'-phosphatase that is also a phosphotransferase, a 1-phosphatase, a long chain acyltransferase with its own acyl carrier protein, and three distinct core glycosyltransferases. Characterization of the R. leguminosarum system should provide insights into the function of lipid A-like molecules, including special roles during symbiosis in plants, and affords the opportunity to create novel lipid A hybrids that may have interesting adjuvant or antagonist activities. Some structural features of R. leguminosarum lipid A are seen in human pathogens. Legionella pneumophila lipid A contains a C28 chain, while Porphyromonas gingivalis and Helicobacter pylori lipid A lack the 4' phosphate. In the coming grant period, the specific aims are: I) cloning of the C28 acyltransferase of R. leguminosarum; II) analysis of the lipid A 4'-phosphatase/phosphotransferase, especially its ability to synthesize PtdIns-4-P; III) determination of the enzymatic basis for proximal unit diversity in R. leguminosarum lipid A; and IV) characterization of enzymes that incorporate the unique inner core sugars of R. leguminosarum LPS.

脂多糖 (LPS) 是一种重要的糖脂，包含革兰氏阴性细菌的外表面，包括共生生物豆根瘤菌。在大肠杆菌中，LPS 的脂质 A 锚是葡萄糖胺的六酰化二糖，在 1 和 4' 位置带有磷酸部分。大肠杆菌生长所需的最低 LPS 由脂质 A 和两种额外的糖组成。新出现的基因组序列表明，在大肠杆菌中产生脂质 A 的酶也存在于大多数其他革兰氏阴性细菌中。脂质 A（通常称为内毒素）也是脂多糖的活性成分，导致革兰氏阴性脓毒症的临床并发症。脂质 A 结构的微小改变会对发病机制产生深远的影响。一些脂质 A 类似物实际上是有效的内毒素拮抗剂。与大肠杆菌相比，豆科细菌 LPS 的脂质 A 和核心结构域的化学结构非常不寻常。 R. leguminosarum 脂质 A 缺乏 1 和 4' 磷酸盐，但在 4' 位置用半乳糖醛酸进行修饰。它被一种特殊的 28 个碳脂肪酸酰化，并含有 2-脱氧-2-氨基葡萄糖酸盐代替近端葡萄糖胺。 R. leguminosarum LPS 的结构表明存在用于产生多种脂质 A 和核心物种的新型酶。现在已经确定，大肠杆菌和豆科细菌中脂质 A 生物合成的前七种酶实际上是相同的。差异出现在途径的后期阶段。迄今为止，被鉴定为豆科植物独有的酶包括 4'-磷酸酶（也是磷酸转移酶）、1-磷酸酶、具有自己的酰基载体蛋白的长链酰基转移酶以及三种不同的核心糖基转移酶。 R. leguminosarum 系统的表征应该可以深入了解类脂质 A 分子的功能，包括植物共生过程中的特殊作用，并提供创建可能具有有趣的佐剂或拮抗剂活性的新型脂质 A 杂合体的机会。豆角杆菌脂质 A 的一些结构特征在人类病原体中可见。嗜肺军团菌脂质A含有C28链，而牙龈卟啉单胞菌和幽门螺杆菌脂质A缺乏4'磷酸。在接下来的资助期内，具体目标是：I）豆科植物C28酰基转移酶的克隆； II)脂质A 4'-磷酸酶/磷酸转移酶的分析，特别是其合成PtdIns-4-P的能力； III)确定豆科菌脂质A中近端单位多样性的酶学基础； IV) 掺入豆科植物 LPS 独特内核糖的酶的表征。