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: 7276369
• 负责人: DEBORAH L PERLSTEIN
• 金额: $ 4.68万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7276369
• 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）：病原菌对我们最有效的药物产生耐药性是一个日益严重的人类健康问题，唯一的治疗方法是开发具有独特作用机制的新型抗生素。转糖基酶是合成细菌生存所必需的细胞壁的细胞外酶，代表了临床使用中多种抗菌药物成功抑制的充满步骤的途径中未充分利用的靶标。新型转糖基酶的鉴定和生化特征将为我们寻找新疗法提供重要信息。项目摘要：由于转糖基酶 (TG) 负责肽聚糖二糖结构单元的聚合，因此它们的活性对于细菌的生存至关重要，但删除枯草芽孢杆菌和粪肠球菌中所有已知的 TG 并不致命。这些生物体以及很可能的许多其他生物体中必定存在一种独特的甘油三酯。此外，缺乏适当的生化工具来剖析“已知”TG 的机制，限制了我们在寻找新抗菌剂时利用这些潜在靶点的能力。 “缺失的”TG 将从枯草芽孢杆菌菌株产生的提取物中纯化，其中所有已知的 TG 均已被删除，并使用 TG 活性测定进行纯化。此外，还将合成并利用基于 TG 底物 Lipid II 的独特光亲和交联试剂来帮助快速识别 TG 候选物。将使用 LC/MS/MS 测序生成 TG 候选基因列表，使用生物信息学分析缩小范围，并通过遗传学和生化实验相结合进行确认。为了了解这种新鉴定的 TG 的机制，将开发一种活性测定方法，包括向每条聚糖链引入单一、独特的放射性探针，并通过尺寸排阻色谱法分离这些链。这种标记方法将使我们首次能够轻松快速地测量 TG 合成的聚糖链的平均大小。此外，我们将能够使用该方法建立更详细的动力学机制来测量聚糖聚合物链引发和链伸长的速率。最后，如果标记脂质 II 是 TG 的底物，那么我们可以使用这种封闭底物来确定延伸方向，因为尚不清楚这些酶是否通过向还原端或非还原端添加新单元来延伸聚糖链。细胞壁形成转糖基酶新家族的第一个成员的发现以及探索TG机制的新工具的开发将是了解这些新抗菌剂尚未充分利用的靶标的重要的第一步。