Activity, control and inhibition of lytic transglycosylases

裂解性转糖基酶的活性、控制和抑制

• 批准号: RGPIN-2016-03965
• 负责人: Clarke, Anthony
• 金额: $ 3.93万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614753
• 关键词: Activity; control; inhibition; lytic; transglycosylases

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.

细菌的基本细胞壁成分肽聚糖（PG）包裹在细胞的细胞质膜上，以维持细胞的结构完整性。尽管如此，新的PG前体结合到生长的PG小囊中，以及后代细胞的分裂，需要溶解（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是内源性细菌酶，专门裂解PG、n -乙酰氨基乙酸和n -乙酰氨基葡萄糖中两种氨基糖之间的连接。它们对细菌至关重要，因为它们是PG合成和细胞分裂，以及分泌/运输系统和附属物的插入所必需的，也可能是孢子复苏所必需的。然而，LTs并没有很好的生物化学特征。我们提出的研究计划建立在我们迄今为止关于LTs的特异性和结构-功能关系的发现之上，我们描述了将探索这些酶在细菌中比以前理解的更广泛分布的可能性的实验。本研究计划的主要目的如下：1.研究目标：开发一种合适的底物用于LTs的测定