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

细菌细胞的形状和大小由肽聚糖囊的形状和大小决定，肽聚糖囊形成细胞渗透稳定性所必需的外骨骼。肽聚糖存在于大多数细菌物种中，其生物合成是广泛使用的、安全有效的抗生素的靶点。虽然许多参与肽聚糖代谢的酶已被表征，但令人惊讶的是，关于它们如何在生长过程中合作以扩大肽聚糖的知之甚少。我们将尝试在大肠杆菌中重组和纯化负责肽聚糖合成的多酶复合物。杆菌应力轴承肽聚糖球囊的扩大需要寡聚化的脂质II前体和其插入到现有的肽聚糖通过转糖基化和转肽。各种合成酶和水解酶参与，在以前的研究中，我们得到的证据表明，酶和其他蛋白质参与功能的多酶复合物的组成部分。我们的目标是从纯化的组分中以其天然形式在体外重建这种复合物。借助功能性肽聚糖合成试验，包括所得肽聚糖的结构分析，我们将研究形成复合物所需的条件和每种蛋白质的作用。特别是，我们将确定的单功能转肽酶的底物特异性，并旨在确定和分析尚未未知的蛋白质-蛋白质相互作用涉及的肽聚糖酶。最终，这些研究将导致对细胞生长过程中肽聚糖层扩大所涉及的分子机制的理解。由于这些反应是细菌特异性的，肽聚糖合成的抑制代表了抗菌化疗的一个很好的目标。了解肽聚糖合成的分子细节对于开发治疗耐药病原体所需的新型抗生素至关重要。

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.