Finding the overlookes transglycosylases in cell wall biosynthesis

寻找细胞壁生物合成中被忽视的转糖基酶

• 批准号: 7382550
• 负责人: DEBORAH L PERLSTEIN
• 金额: $ 4.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7382550
• 关键词: Address; Anabolism; Anti-Bacterial Agents; Antibiotics; Bacillus subtilis; Bacteria; Bacterial Physiology; Biochemical; Biochemical Genetics; Biochemistry; Bioinformatics; Biological Assay; Candidate Disease Gene; Carbohydrates; Cell Wall; Chemicals; Clinical; Condition; Cross-Linking Reagents; Development; Disaccharides; Enterococcus faecalis; Enzymes; Family; Galactose; Galactosyltransferases; Gene Deletion; Genes; Genetic; Health; Human; Hydrophobic Interactions; Hydroxyl Radical; Investigation; Ion Exchange; Kinetics; Label; Lipids; Measures; Membrane; Methods; Molecular Sieve Chromatography; Organism; Paper Chromatography; Pathway interactions; Peptidoglycan; Pharmaceutical Preparations; Polymers; Polysaccharides; Product Labeling; Proteins; Radioactive; Rate; Resistance; Role; Techniques; Time; base; crosslink; extracellular; member; novel therapeutics; pathogenic bacteria; polymerization; research study; size; tool

DESCRIPTION (provided by applicant): The resistance of pathogenic bacteria to our most effective drugs is an ever-growing human health problem for which the only cure is the development of new antibiotics functioning with unique mechanisms of action. The transglycosylases, extracellular enzymes that synthesize the cell wall essential for the bacterial survival, represent an underexploited target in a pathway full of steps successfully inhibited by numerous antibacterials in clinical use. The identification and biochemical characterization of a new kind of transglycosylase will provide vital information that could be exploited in our search for novel therapeutics. Project Summary: Since the transglycosylases (TGs) are responsible for polymerization of the disaccharide building blocks of peptidoglycan, their activity is essential for bacterial survival and yet, deletion of all known TGs in B. subtilis and E. faecalis is not lethal. A unique kind of TG must exist in these and, most likely, many other organisms. Furthermore, the lack of appropriate biochemical tools for dissecting the mechanism of the "known" TGs has limited our ability to exploit these potential targets in the search for new antibacterial agents. The "missing" TG will be purified from extracts generated from a B. subtilis strain in which all known TGs have been deleted, using a TG activity assay to follow purification. Additionally, a unique photoaffinity cross-linking reagent based on the TG substrate, Lipid II, will be synthesized und utilized to help rapidly identify TG candidates. A list of TG candidate genes will be generated using LC/MS/MS sequencing, narrowed using a bioinformatics analysis, and confirmed by a combination of genetics and biochemical experiments. To understand the mechanism of this newly identified TG, an activity assay will be developed involving introduction of a single, unique radioactive probe to each glycan strand and separation of these strands by size-exclusion chromatography. This labeling method will allow us to, for the first time, easily and rapidly measure the average size of the glycan strands synthesized by TGs. In addition, we will be able to build a more detailed kinetic mechanism using this method to measure the rates of glycan polymer chain initiation and chain elongation. Finally, if labeled-Lipid II is a substrate for the TGs, then we can use this blocked substrate to determine the direction of elongation because it is unknown if these enzymes extend the glycan chain through addition of new units to the reducing or non-reducing end. The discovery of the first member of a new family of cell wall forming transglycosylases and the development of new tools to probe the TG mechanism will be important first steps towards understanding these underexploited targets for new antibacterial agents.

描述（由申请人提供）：致病细菌对我们最有效的药物的抗性是一个不断增长的人类健康问题，唯一的治愈方法是开发新的抗生素具有独特的作用机理。在临床用途中，在充满步骤成功抑制的途径中，构成细菌生存必不可少的细胞壁的抗糖基酶，是合成细菌生存必不可少的细胞壁的细胞外酶。一种新型的经糖基酶的识别和生化表征将提供重要的信息，在我们寻找新型治疗剂时可以利用这些信息。项目摘要：由于晶糖基酶（TGS）负责肽聚糖的二糖构建块的聚合，因此它们的活性对于细菌存活至关重要，但是，在枯草芽孢杆菌和粪肠中所有已知的TGS的缺失并不是杀菌。这些独特的TG必须存在于其中，而且很可能是许多其他生物。此外，缺乏剖析“已知” TGS机制的适当生化工具，限制了我们利用这些潜在靶标在寻找新抗菌剂时的能力。使用TG活性测定法以遵循纯化，将从枯草芽孢杆菌菌株中产生的提取物中纯化“缺失” TG。此外，将合成基于TG底物脂质II的独特光亲和力交联试剂，并将合成，以帮助快速识别TG候选物。使用LC/MS/MS测序将生成TG候选基因列表，并使用生物信息学分析范围缩小，并通过遗传学和生物化学实验的组合确认。为了了解这种新鉴定的TG的机制，将开发一种活性测定，涉及向每个聚糖链引入单个独特的放射性探针，并通过尺寸排斥色谱法对这些链的分离进行分离。这种标签方法将使我们首次轻松，快速地测量由TG合成的聚糖链的平均大小。此外，我们将能够使用这种方法来构建更详细的动力学机制，以衡量聚糖聚合物链的启动和链伸长的速率。最后，如果标记的脂质II是TGS的底物，那么我们可以使用此阻断的底物来确定伸长方向，因为这些酶是否通过将新单元添加到还原或非还原端来扩展了聚糖链。发现新的细胞壁家族的第一个成员，形成了经糖基化酶，并开发了探测TG机制的新工具，将是理解这些新抗菌剂的这些不宽容目标的重要第一步。