Biosynthesis of Polysaccharides

描述（由申请人提供）：碳水化合物在自然界中普遍存在，并在各种重要的生物过程中发挥重要作用。与核酸和蛋白质生物合成的分子机制的深入理解相反，多糖生物合成的理解很少。尽管多糖具有显著的结构多样性，但基于先前广泛的遗传研究，仅利用了三种主要的延伸/聚合机制，即wzy依赖性、ABC转运蛋白依赖性和淀粉酶依赖性。其中，wzy依赖性途径是自然界中应用最广泛的途径。它与多种杂多糖如0-多糖、荚膜多糖、胞外多糖、结肠酸和肠细菌共同抗原的生物合成有关。目前wzy依赖性途径的工作模型是通过特异性糖基转移酶的顺序作用在细胞质中合成单个重复寡糖单元。然后将重复单元转运到膜的周质侧，在那里它通过聚合酶Wzy聚合成多糖。聚合物的链长由Wzz蛋白调节。到目前为止，除了基因敲除实验之外，这种粗略的生物合成图景从未在体外进行过实验测试。大多数的关键酶在假设的途径从来没有生化特性和他们的作用机制（特别是O-多糖聚合，调节和运输）是相当投机。在过去的三年里，利用E. coli O 86 O-多糖作为模型系统，我们在理解多糖生物合成方面取得了重大进展。在这一拟议的研究期间，我们计划重点关注以下三个具体目标：目标1。O-单元-PP-脂质底物/类似物的化学-酶促合成。这些化学定义的底物将用于探测Wzy聚合反应机理。目标2.聚合酶Wzy的研究。本节包括体外试验的开发，以获得可靠的动力学和脂质特异性和链延伸方向的研究。目标3。研究Wzz和探索链长调节。本节包括使用一系列生物化学和生物物理方法研究Wzy-Wzz相互作用、Wzz-多糖相互作用和Wzz寡聚状态。总之，这个长期的研究计划将揭示复杂碳水化合物聚合物的详细生物合成机制。对多糖生物合成的深入了解将对新型抗生素、疫苗、免疫抑制剂和抗炎剂的研究和开发产生相当大的影响。 公共卫生相关性：许多致病菌被由结构多样的高分子量多糖组装而成的荚膜结构包围。这些细胞表面多糖在介导细菌细胞与其环境之间的相互作用中起重要作用，并且被认为是重要的致病因子。因此，从分子水平上了解多糖在细胞中的生物合成是一个具有重要医学意义的重要生物学问题。例如，它可以为开发针对新出现的耐药性问题的新抗生素提供新的目标;检测新病原菌的新方法;生产多糖疫苗，免疫抑制剂和抗炎剂的新策略。

DESCRIPTION (provided by applicant): Carbohydrates are ubiquitous in nature and play an essential role in a variety of important biological processes. In contrast to the in-depth understanding of the molecular mechanisms of nucleic acid and protein biosynthesis, polysaccharide biosynthesis is poorly understood. In spite of the remarkable structural diversity of polysaccharides, only three major elongation/polymerization mechanisms, namely wzy- dependent, ABC-transporter dependent and synthase-dependent, are utilized based on previous extensive genetic studies. Among them, the wzy-dependent pathway is the most widely used in nature. It has been implicated in the biosynthesis of a variety of hetero-polysaccharides such as O-polysaccharides, capsular polysaccharides, exo- polysaccharides, colonic acids and enterobacterial common antigens. The current working model for the wzy-dependent pathway is that the individual repeating oligosaccharide unit is synthesized in the cytoplasm by the sequential action of specific glycosyltransferases. The repeating unit is then transported to the periplasmic side of the membrane where it is polymerized into a polysaccharide by the polymerase Wzy. The chain length of the polymer is regulated by the Wzz protein. So far, with the exception of gene knockout experiments, this rough biosynthetic picture has never been experimentally tested in vitro. Most of the critical enzymes in the hypothesized pathway have never been biochemically characterized and their mechanisms of action (especially for the O-polysaccharide polymerization, regulation and transport) are rather speculative. In the past three years, using E. coli O86 O-polysaccharide as a model system, we have made significant progress towards understanding polysaccharide biosynthesis. In this proposed research period, we plan to focus on the following three specific aims: Aim 1. Chemo-enzymatic synthesis of O-unit-PP-lipid substrates/analogs. These chemically defined substrates will be used for probing Wzy polymerization reaction mechanism. Aim 2. Investigation of polymerase Wzy. This section includes development of in vitro assays to obtain reliable kinetics and investigation of lipid specificity and chain elongation direction. Aim 3. Investigation of Wzz and probing chain length regulation. This section includes the investigation of Wzy-Wzz interaction, Wzz-polysaccharide interaction and Wzz oligomeric states using a series of biochemical and biophysical approaches. In summary, this long-term research program will uncover the detailed biosynthetic mechanism of complex carbohydrate polymers. An in-depth understanding of biosynthesis of polysaccharides will have a considerable impact on research and development of novel antibiotics, vaccines, immuno-suppressors, and anti-inflammatory agents. PUBLIC HEALTH RELEVANCE: Many pathogenic bacteria species are surrounded by a capsular structure that is assembled from structurally diverse, high molecular weight polysaccharides. These cell surface polysaccharides play an essential role in mediating interactions between bacterial cells and their environment, and are recognized as an important pathogenic factor. Thus understanding how the polysaccharides are biosynthesized in the cell in molecular level is a fundamentally important biological question with significant medical implications. For example, it can provide new targets for the development of new antibiotics against emerging drug resistance problems; new methods to detect new pathogenic bacteria; new strategies to generate polysaccharide-based vaccines, immuno-suppressors and anti-inflammatory agents.