Biosynthesis of Polysaccharides

描述（由申请人提供）：碳水化合物在自然界中无处不在，在多种重要的生物过程中起着至关重要的作用。与对核酸和蛋白质生物合成的分子机制的深入了解相比，对多糖生物合成的了解甚少。尽管多糖具有显著的结构多样性，但基于以往广泛的遗传研究，仅利用了三种主要的延伸/聚合机制，即依赖于wzy，依赖于abc转运体和依赖于合成酶。其中，wzy依赖通路在自然界中应用最为广泛。它参与了多种异多糖的生物合成，如o型多糖、荚膜多糖、外多糖、结肠酸和肠杆菌常见抗原。目前wzy依赖途径的工作模型是，通过特定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，重复单元被运送到膜的质周侧，在那里它被聚合酶Wzy聚合成多糖。聚合物的链长是由Wzz蛋白调控的。到目前为止，除了基因敲除实验外，这种粗略的生物合成图从未在体外进行过实验测试。在假设的途径中，大多数关键酶从未被生物化学表征，其作用机制（特别是对o -多糖的聚合、调节和运输）相当推测。在过去的三年中，我们以大肠杆菌O86 o -多糖为模型系统，在了解多糖的生物合成方面取得了重大进展。在这个拟议的研究期间，我们计划重点关注以下三个具体目标：目标1。o- unit- pp -脂质底物/类似物的化学酶合成。这些化学定义的底物将用于探究Wzy聚合反应机理。目标2。聚合酶Wzy的研究。本节包括体外测定的发展，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。Wzz的研究和探索链长调节。本节包括使用一系列生化和生物物理方法研究Wzz- Wzz相互作用、Wzz-多糖相互作用和Wzz寡聚物状态。总之，这项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。深入了解多糖的生物合成将对新型抗生素、疫苗、免疫抑制剂和抗炎剂的研究和开发产生重大影响。

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.