Biosynthesis of Polysaccharides

多糖的生物合成

• 批准号: 7906823
• 负责人: Peng George Wang
• 金额: $ 31万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-03 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7906823
• 关键词: ATP-Binding Cassette Transporters; Acids; Anabolism; Anti-Inflammatory Agents; Antibiotics; Biochemical; Biological; Biological Models; Biological Process; Carbohydrates; Cell Communication; Cell surface; Cells; Complex; Cytoplasm; Data; Development; Drug resistance; Embryonic Development; Environment; Enzymes; Escherichia coli; Genetic; Immune response; Individual; Investigation; Kinetics; Laboratories; Length; Life; Lipids; Mediating; Mediation; Medical; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Molecular Weight; Nature; Nucleic Acids; Oligosaccharides; Organism; Pathway interactions; Play; Polymerase; Polymers; Polysaccharides; Protein Biosynthesis; Proteins; Reaction; Regulation; Research; Role; Series; Side; Signal Transduction; Solutions; Specificity; Structure; Time; Vaccines; Work; analog; base; colanic acid; design; enterobacterial common antigen; glycosyltransferase; in vitro Assay; in vitro testing; insight; knockout gene; lipid structure; macromolecule; novel; pathogenic bacteria; periplasm; polymerization; preference; programs; public health relevance; research and development; research study; sugar

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.

描述（由申请人提供）：碳水化合物本质上是普遍存在的，并且在各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反，多糖生物合成知之甚少。尽管多糖的显着结构多样性，但仅基于先前的广泛遗传研究，仅利用了三种主要的伸长/聚合机制，即Wzy-Wzy-依赖性，ABC转运蛋白依赖性和合成酶依赖性。其中，WZY依赖性途径是自然界中最广泛使用的途径。它与多种杂多糖，诸如O-多糖，囊囊多糖，外可多糖，结肠酸和肠细菌常见抗原等多种异质 - 甲基糖浆的生物合成有关。 WZY依赖性途径的当前工作模型是，单个重复寡糖单位是通过特定糖基转移酶的顺序作用在细胞质中合成的。然后将重复单元转移到膜的周质侧，在该膜中通过聚合酶Wzy将其聚合到多糖中。聚合物的链长由WZZ蛋白调节。到目前为止，除了基因敲除实验外，这种粗糙的生物合成图片从未在体外进行实验测试。假设途径中的大多数关键酶从未在生化上表征，其作用机理（尤其是对于O-碳酸盐聚合，调节和运输）是相当投机性的。在过去的三年中，使用大肠杆菌O86 O-杆糖作为模型系统，我们在理解多糖生物合成方面取得了重大进展。在这个拟议的研究期间，我们计划关注以下三个特定目的：目标1。O-UNIT-PPP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测WZY聚合反应机制。目标2。聚合酶WZY的研究。本节包括开发体外测定，以获得可靠的动力学以及研究脂质特异性和链伸长方向的研究。目标3。调查WZZ和探测链长度调节。本节包括使用一系列生化和生物物理方法研究WZY-WZZ相互作用，WZZ-碳相互作用和WZZ低聚物状态的研究。总而言之，该长期研究计划将发现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，免疫抑制剂和抗炎药的研究和开发产生相当大的影响。 公共卫生相关性：许多致病性细菌被囊状结构包围，该结构由结构多样的高分子重量多糖组装而成。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着至关重要的作用，并被认为是重要的致病因素。因此，了解多糖在分子水平中如何在细胞中生物合成是具有重要医学意义的根本重要生物学问题。例如，它可以为开发新的抗生素针对新兴耐药性问题提供新的靶标。检测新的致病细菌的新方法；生成基于多糖的疫苗，免疫抑制剂和抗炎药的新策略。