Activity, control and inhibition of lytic transglycosylases

细菌肽聚糖（PG）的基本细胞壁组分包围细胞的细胞质膜以维持其结构完整性。 尽管如此，新的PG前体进入生长的PG球囊，连同后代细胞的分裂，需要溶解（自溶）酶的作用。 由细菌产生的一类自溶酶（自溶素）是溶解性转糖基酶（LT）。 LT是内源性细菌酶，其特异性裂解PG中的两种氨基糖之间的连接，N-乙酰胞壁酸和N-乙酰葡糖胺。 它们对细菌是必不可少的，因为它们是PG合成和细胞分裂所必需的，以及分泌/运输系统和附属物的插入，并且可能是孢子复苏所必需的。 然而，LT没有很好地进行生物化学表征。提出的研究计划建立在我们迄今为止对LT的特异性和结构-功能关系的研究结果的基础上，我们描述了一些实验，这些实验将探索这些酶在细菌中比以前所了解的更广泛分布的可能性。因此，本研究计划的主要目的是：1。开发用于LT测定的合适底物 *2。使用这种新底物对大肠杆菌和铜绿假单胞菌的LT进行生化和动力学分析，并发现新的LT *3。研究溶菌酶的蛋白质抑制剂对LT的抑制 *4。通过蛋白质-蛋白质相互作用研究LT的生理控制。*这些研究将涉及微生物、分子生物学和分析生化技术的组合，包括电子显微镜、定点诱变、酶动力学分析以及通过表面等离子体共振（SPR）和SUPREX技术分析LT与小分子和其他蛋白质的结合特性。我们还提出了实验来解决的问题，为什么LT执行转糖基化反应，而不是简单的水解催化的胞壁酶（溶菌酶）在真核生物中发现的先天免疫系统的组成部分，为什么LT局限于细菌和噬菌体。因此，我们计划测试我们的假设，即LT在N-乙酰胞壁酰反应产物中保留氨基糖之间的裂解糖基键作为1，6-脱水键，用于其随后与其他GlcNAc残基的再连接。我们进一步假设LT催化了这种逆反应。

The essential cell wall component of bacteria peptidoglycan (PG) encloses the cytoplasmic membrane of cells to maintain their structural integrity. Nonetheless, the incorporation of new PG precursors into the growing PG sacculus, together with the division of progeny cells, requires the action of lytic (autolytic) enzymes. One class of autolytic enzymes (autolysins) produced by bacteria are the lytic transglycosylases (LTs). The LTs are endogenous bacterial enzymes that specifically lyse the linkage between the two aminosugars in PG, N-acetylmuramic acid and N-acetylglucosamine. They are essential to bacteria because they are required for PG synthesis and cell division, as well as insertion of secretion/transport systems and appendages, and possibly for spore resuscitation. However, the LTs are not well characterized biochemically. The research program proposed builds on our findings to date on the specificity and structure-function relationship of the LTs, and we describe experiments that will explore the possibility that these enzymes are more wide-spread in bacteria than previously understood. The major aims of this research program are thus to:1. Develop a suitable substrate for the assay of LTs ***2. Use this new substrate for the biochemical and kinetic analyses of LTs from Escherichia coli and Pseudomonas aeruginosa and for the discovery of new LTs***3. Investigate the inhibition of the LTs by the proteinaceous inhibitors of lysozyme***4. Investigate the physiological control of the LTs through protein-protein interactions.***These studies will involve a combination of microbiological, molecular biological,and analytical biochemical techniques, including electron microscopy, site-directed mutagenesis, enzyme kinetic analyses, and analysis of the binding properties of LTs to both small molecules and other proteins by both surface plasmon resonnance (SPR) and SUPREX technologies. We also propose experiments to address the question of why the LTs perform a transglycosylation reaction rather than the simple hydrolysis catalysed by the muramidases (lysozymes) found in eukaryotic organisms as components of innate immune systems, and why LTs are confined to bacteria and phage. Thus, we plan to test our hypothesis that the LTs retain the cleaved glycosyl linkage between the aminosugars as 1,6-anhydro bonds in N-acetylmuramyl reaction products for their subsequent re-ligation to other GlcNAc residues. We further postulate that the LTs catalyse this reverse reaction.********