Finding the overlookes transglycosylases in cell wall biosynthesis

描述(申请人提供)：病原菌对我们最有效的药物的耐药性是一个日益严重的人类健康问题，唯一的解决办法是开发具有独特作用机制的新抗生素。转糖基酶是一种合成细菌生存所必需的细胞壁的胞外酶，在临床上被许多抗菌药物成功抑制的一系列步骤中，它是一个未被开发的靶点。一种新的转糖基酶的鉴定和生化特性将为我们寻找新的治疗方法提供重要的信息。项目摘要：由于转糖基酶(TGS)负责肽聚糖的二糖组分的聚合，它们的活性对细菌的生存是必不可少的，然而，在枯草杆菌和粪肠球菌中所有已知的TGS的缺失并不是致命的。一种独特的甘油三酯必须存在于这些生物中，而且很可能存在于许多其他生物中。此外，缺乏适当的生化工具来剖析已知的TGS的机制，限制了我们在寻找新的抗菌剂时利用这些潜在靶点的能力。“缺失的”TG将从枯草杆菌菌株产生的提取物中提纯，在该菌株中所有已知的TG都已被删除，使用TG活性分析来进行纯化。此外，一种基于TG底物的独特的光亲和交联剂Lipid II将被合成并用于帮助快速识别TG候选对象。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的方式进行确认。为了了解这种新发现的甘油三酯的作用机制，将开发一种活性分析方法，在每一条糖链上引入一个唯一的放射性探针，并通过尺寸排除层析分离这些链。这种标记方法将使我们第一次能够容易和快速地测量由TGS合成的糖链的平均尺寸。此外，我们将能够利用这种方法建立更详细的动力学机制来测量葡聚糖高聚物链的起始率和链长。最后，如果标记的Lipid II是TGS的底物，那么我们可以使用这个被阻止的底物来确定延伸的方向，因为尚不清楚这些酶是否通过在还原或非还原末端添加新单元来延长糖链。形成转糖基酶的细胞壁新家族的第一个成员的发现和探索TG机制的新工具的开发将是理解这些未被开发的新抗菌药物靶点的重要的第一步。

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.