Activity, control and inhibition of lytic transglycosylases

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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.