CLONING OF NOVEL ENZYMES THAT MODIFY LIPID A

修饰脂质 A 的新型酶的克隆

• 批准号: 6136267
• 负责人: Michael Stephen Trent
• 金额: $ 3.24万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6136267
• 关键词: Salmonella typhimurium; acylation; amidases; arabinose; bacterial genetics; bacterial proteins; endotoxins; lipid biosynthesis; lipopolysaccharides; mass spectrometry; microorganism culture; microorganism metabolism; molecular cloning; nuclear magnetic resonance spectroscopy; pentosyltransferase; virulence

The outer leaflet of the outer membranes of Gram-negative bacteria is almost entirely composed of lipopolysaccharide (LPS) and acts as an efficient permeability barrier against antibacterial agents. Lipid A (endotoxin) functions as the hydrophobic anchor of LPS and is the bioactive component of the molecule leading to Gram-negative septic shock and death. The enzymatic machinery required for lipid A biosynthesis is well understood, however, the enzymes responsible for certain regulated modifications of lipid A remain unknown. These modifications include the addition of 4-aminoarabinose, phosphoethanoiamine, palmitate, 2- hydroxymyristate, phosphate, and the removal of specific fatty acyl chains. It is now known that some of these modifications are important for survival of the bacterium from the host innate immune system. Therefore, understanding the enzymology of these modifications will not only facilitate the understanding of lipid A biosynthesis but will lay the foundation of new molecular insights into pathogenesis. The current study focuses on (I) the characterization of enzymes involved in the addition of 4-aminoarabinose and deacylation of lipid A; (II) structural analysis of the enzymatic products and analysis of their endotoxic activities; and (III) the cloning of these novel genes.

革兰氏阴性菌外膜的外小叶几乎完全由脂多糖（LPS）组成，并作为抗抗菌剂的有效渗透屏障。脂质A（内毒素）作为LPS的疏水性锚发挥作用，并且是导致革兰氏阴性脓毒性休克和死亡的分子的生物活性组分。脂质A生物合成所需的酶机制是众所周知的，然而，负责脂质A的某些调节修饰的酶仍然未知。这些修饰包括添加4-氨基阿拉伯糖、磷酸乙醇胺、棕榈酸酯、2-羟基肉豆蔻酸酯、磷酸酯和去除特定的脂肪酰基链。现在已知这些修饰中的一些对于细菌从宿主先天免疫系统中存活是重要的。因此，了解这些修饰的酶学不仅有助于了解脂质A的生物合成，而且将为发病机制的新分子见解奠定基础。目前的研究重点是（I）参与添加4-氨基阿拉伯糖和脂质A脱酰化的酶的表征;（II）酶产物的结构分析及其内毒素活性分析;和（III）这些新基因的克隆。