Reconstitution and functional analysis of multi-enzyme complexes involved in Escherichia coli peptidoglycan synthesis

点击翻译按钮获取中文摘要

The shape and size of a bacterial cell is determined by the shape and size of the peptidoglycan sacculus, which forms an exoskeleton that is essential for the osmotic stability of the cell. Peptidoglycan is present in most bacterial species and its biosynthesis is the target of widely used, safe and effective antibiotics. Although many of the enzymes involved in peptidoglycan metabolism have been characterised, surprisingly little is known about how they co-operate to enlarge the peptidoglycan during growth. We will attempt to reconstitute and characterise the multi-enzyme complexes responsible for peptidoglycan synthesis in E. coli. The enlargement of the stress-bearing peptidoglycan sacculus requires the oligomerization of the lipid II precursor and its insertion into existing peptidoglycan by transglycosylation and transpeptidation. Various synthetic and hydrolytic enzymes are involved, and in previous studies we obtained evidence that the synthases and other proteins involved function as components of multi-enzyme complexes. We aim to reconstitute such complexes in vitro in their native forms from the purified components. With the help of a functional peptidoglycan synthesis assay involving the structural analysis of the resulting peptidoglycan, we will study the conditions required for the formation of complexes and the role of each protein. In particular, we will determine the substrate specificity of the monofunctional transpeptidases, and aim to identify and characterise as yet unknown protein-protein interactions involving the peptidoglycan synthases. Ultimately these studies will lead to an understanding of the molecular mechanisms involved in the enlargement of the peptidoglycan layer during cell growth. Because these reactions are specific to bacteria, the inhibition of the peptidoglycan synthesis represents an excellent target for antimicrobial chemotherapy. Knowledge of the molecular details of peptidoglycan synthesis will be crucial for the development of novel antibiotics required to treat drug-resistant pathogens.