BIOSYNTHESIS OF MEMBRANE GLYCOLIPIDS IN RHIZOBIUM

脂多糖（lps）是组成革兰氏阴性菌（包括共生生物豆科根瘤菌）外表面的显著糖脂。在大肠杆菌中，脂质A锚点是葡萄糖胺的六酰化双糖，在1和4'位置上含有磷酸基团。大肠杆菌生长所需的最低脂多糖包括脂质a和两种额外的糖。新出现的基因组序列表明，大肠杆菌中制造脂质A的酶存在于大多数其他革兰氏阴性菌中。脂质A（通常称为内毒素）也是引起革兰氏阴性脓毒症临床并发症的脂多糖的活性成分。脂质A结构的微小改变可对发病机制产生深远影响。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，豆科植物脂质A和核心结构域的化学结构非常不寻常。豆科植物脂质A缺乏和4′磷酸，但在4′位被半乳糖醛酸修饰。它与一种特殊的28碳脂肪酸酰化，并含有2-脱氧-2-氨基葡萄糖酸盐代替近端氨基葡萄糖。豆科植物脂多糖的结构表明存在生成多种脂质A和核心种的新型酶。现在已经确定，在大肠杆菌和豆科致病菌中，脂质A生物合成的前7种酶实际上是相同的。差异出现在通路的后期阶段。迄今为止，已知的豆科植物特有的酶包括一个4'-磷酸酶（也是一个磷酸转移酶），一个1-磷酸酶，一个带有酰基载体蛋白的长链酰基转移酶，以及三个不同的核心糖基转移酶。对R. leguminosarum系统的表征有助于深入了解脂质A样分子的功能，包括在植物共生过程中的特殊作用，并为创造可能具有有趣的佐剂或拮抗剂活性的新型脂质A杂交种提供机会。豆科致病菌脂质A的一些结构特征在人类致病菌中可见。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：1)克隆豆科植物C28酰基转移酶；II)脂质4a '-磷酸酶/磷酸转移酶的分析，特别是其合成PtdIns-4-P的能力；III)确定豆豉脂质A近端单位多样性的酶基础；IV)结合蚕豆多糖独特内核糖的酶的表征。

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.