The function of peptidoglycan-active enzymes

这个建议是我继续研究碳水化合物活性酶的结构和功能关系的计划。在过去的三十多年里，我的实验室已经发展了专业知识，在细菌细胞壁杂聚物肽聚糖（PG）的代谢和植物细胞壁聚合物纤维素和木聚糖的生物降解所涉及的酶的研究。该研究计划建立在我们迄今为止关于裂解性转糖基酶（LT）的特异性、结构和功能关系以及酶促控制的研究结果的基础上，LT是在PG的生物合成、成熟和周转中起重要作用的内源性细菌自溶素。PG裂解导致细胞破裂和死亡），这些酶必须在酶和/或底物水平上进行控制。我们的研究表明，许多细菌通过其底物PG的O-乙酰化来实现这种控制。我们发现并继续表征进行这种修饰并去除控制O-乙酰化以允许继续PG代谢的酶。然而，大量革兰氏阴性细菌（分别包括大肠杆菌和铜绿假单胞菌以及相关物种）缺乏这种修饰其PG的能力。相反，我们发现来自铜绿假单胞菌的LT被“脊椎动物溶菌酶抑制剂”（Ivy）有效抑制，这种蛋白质被认为可以保护革兰氏阴性菌免受宿主先天免疫系统溶菌酶的防御作用。此外，我们发现了一个直接和严格的相关性，这些细菌产生常春藤样蛋白质与那些不O乙酰化他们的PG。因此，我们假设，真正的生理功能常春藤及其直系同源物和旁系同源物是控制内源性自溶素的生产细菌。因此，本研究计划的总体目标是通过测试两个特定的假设，充分表征LT的作用和活动。首先，LT活性是由已发现的溶菌酶的各种蛋白质抑制剂控制的。我们还建议解决的根本问题，为什么LT执行转糖基化反应产生的分子内1，6-脱水键胞壁酰产品，而不是一个简单的水解，作为先天免疫系统的组成部分，在真核生物中发现的胞壁酶（溶菌酶）催化？在这方面，我们将测试我们的假设，即LT保留切割的α-（1，4）糖基键作为1，6脱水键，用于随后重新连接到适当的受体（例如，相邻PG链上的GlcNAc残基或跨壁结构的蛋白质）。对这些基本问题的深入了解将大大加深我们对LT在细菌细胞壁重要成分PG代谢中的作用和功能的理解。

This proposal concerns the continuation of my research program that investigates the structure and function relationship of carbohydrate-active enzymes. Over the past thirty plus years, my lab has developed expertise in the study of enzymes involved in the metabolism of the bacterial cell wall heteropolymer peptidoglycan (PG) and the biodegradation of the plant cell wall polymers cellulose and xylan. The research program builds on our findings to date on the specificity, structure and function relationship, and enzymatic control of the lytic transglyosylases (LTs), endogenous bacterial autolysins that play essential roles in the biosynthesis, maturation, and turnover of PG. Given their potentially catastrophic autolytic activity (i.e., lysis of PG leading to cell rupture and death), these enzymes have to be controlled at the enzyme and/or substrate level. Our research has shown that many bacteria accomplish this control by the O-acetylation of their substrate PG. We discovered and continue to characterize the enzymes that both perform this modification and remove the controlling O-acetylation to permit continued PG metabolism. However, a large number of Gram-negative bacteria  (including Escherichia coli and Pseudomonas aeruginosa and related species, respectively) lack this ability to modify their PGs. Instead, we found that an LT from P. aeruginosa is efficiently inhibited by "inhibitors of vertebrate lysozyme" (Ivy), proteins thought to provide protection to Gram-negative bacteria from the defensive action of the lysozymes of host innate immune systems. Moreover, we found a direct and strict correlation between those bacteria that produce Ivy like proteins with those that do not O acetylate their PG. Hence, we have postulated that the true physiological function of Ivy and its orthologs and paralogs is to control the endogenous autolysins of the producing bacteria. Thus, the overarching aims of this research program are to fully characterize the role and activity of the LTs by testing two specific hypotheses. The first is that LT activity is controlled by the various proteinaceous inhibitors of lysozyme that have been discovered. We also propose to address the fundamental question of why the LTs perform a transglycosylation reaction generating an intramolecular 1,6-anhydro bond in muramoyl products rather than a simple hydrolysis as catalyzed by the muramidases (lysozymes) found in eukaryotic organisms as components of innate immune systems? In this regard, we will test our hypothesis that the LTs retain the cleaved ß-(1,4) glycosyl linkage as the 1,6 anhydro bond for subsequent re-ligation to appropriate acceptors (eg., GlcNAc residues on neighboring PG strands or proteins of wall-spanning structures) during the process of PG re-modelling. Insight to these fundamental questions will greatly further our understanding of the role and function of the LTs in the metabolism of the essential component of bacterial cell walls, PG.