Activity, control and inhibition of lytic transglycosylases

细菌肽聚糖(PG)是细菌的基本细胞壁成分，它包裹在细胞的细胞膜上，以维持细胞结构的完整性。然而，将新的PG前体结合到生长中的PG球囊中，以及子代细胞的分裂，需要裂解(自溶)酶的作用。细菌产生的一类自溶酶(自溶素)是裂解转糖基酶(LTS)。LTS是一种内源性细菌酶，能特异性地分解PG，N-乙酰胞壁酸和N-乙酰氨基葡萄糖中两种氨基糖之间的连接。它们对细菌来说是必不可少的，因为它们是PG合成和细胞分裂所必需的，也是分泌/运输系统和附属物插入所必需的，可能也是孢子复苏所必需的。然而，LTS的生化特性并不是很好。提出的研究计划建立在我们迄今对LTS的特异性和结构功能关系的发现的基础上，我们描述了一些实验，这些实验将探索这些酶在细菌中比之前所知的更广泛分布的可能性。因此，本研究计划的主要目的是：1.开发一种适合测定LTS的底物。2.利用这一新的底物对来自大肠杆菌和铜绿假单胞菌的LTS进行生化和动力学分析，并发现新的LTS。4.研究溶菌酶的蛋白质类抑制剂对LTS的抑制作用。通过蛋白质-蛋白质相互作用来研究LTS的生理调控。*这些研究将结合微生物学、分子生物学和生化分析技术，包括电子显微镜、定点突变、酶动力学分析、以及利用表面等离子激元共振(SPR)和SUPREX技术分析LTS与小分子和其他蛋白质的结合特性。我们还建议进行实验，以解决为什么LTS执行转糖基化反应，而不是由真核生物中发现的溶菌酶(溶菌酶)作为先天性免疫系统的组成部分所催化的简单水解，以及为什么LTS仅限于细菌和噬菌体。因此，我们计划检验我们的假设，即LT在N-乙酰胞壁酰化反应产物中以1，6-脱水键的形式保留氨基糖之间的糖基键，以便随后与其他GlcNAc残基重新连接。我们进一步假设LTS催化了这一逆反应。

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.********