Phosphotransferases in bacterial cell wall biosynthesis

细胞壁是细菌两个主要分支之一的一个重要的、有时被忽视的关键方面，称为革兰氏阳性菌。如果没有细胞壁，革兰氏阳性细菌细胞将严重受损，细胞中的可溶性物质将穿透包裹细胞的相当脆弱的单一脂膜。细胞就会死亡。这些生物的细胞壁由两个主要成分组成，一个网状结构，由交联链的肽聚糖组成，它与长的聚合物碳水化合物如磷壁酸和胶囊相连。肽聚糖和磷壁酸都发挥着关键作用--肽聚糖提供必要的物理保护，使其免受细胞渗透压力的影响，其合成与细胞的生长相协调，从而使肽聚糖不受限制。磷壁酸是细菌与其周围环境相互作用所必需的，也是调节细胞表面其他酶活动所必需的，并有助于维持细胞的整体形状。通常需要胶囊来保护病原体免受免疫系统组件的攻击。磷壁酸、胶囊多糖和肽聚糖是由一系列称为酶的特定生物催化剂以多个独立的步骤合成的。磷壁酸和胶囊多糖是在细胞内合成的，而肽聚糖是在细胞外合成的。脂质连接的磷壁酸前体被跨膜运输并转移到肽聚糖上，尽管这一转移步骤发生在肽聚糖合成的确切阶段尚不清楚。只有这种主要的阴离子聚合物(壁磷壁酸和胶囊)附着到肽聚糖上，才能形成细菌生活方式所必需的最终的、有功能的细胞壁。我们已经发现了一种名为LCP的酶家族，它执行这一转移步骤，在这个建议中，我们试图了解其生化特性的分子基础。具体地说，我们将研究这些酶催化的生化反应，并确定这些酶与反应组分形成的复合体的高分辨三维结构。我们还将开发在该途径中已知的酶的催化下合成磷壁酸构筑块，与我们迄今使用的商业可用成分相比，它将更适合于这些蛋白质的生物化学研究。

The cell wall is an important, and sometimes overlooked, key facet of one of two major branches of bacteria, called the Gram-positives. Without the cell wall, the Gram-positive bacterial cell would become seriously compromised and the soluble contents of the cell would burst through the rather fragile single lipid membrane that encompasses the cell. The cell would die. The cell wall of these organisms comprises two major components, a mesh-like structure of cross-linked strands of peptidoglycan, which becomes attached to long, polymeric carbohydrates, such as the teichoic acids and the capsule. Both peptidoglycan and teichoic acid play key roles - peptidoglycan provides the necessary physical protection from the osmotic pressure of the cell, and its synthesis is co-ordinated with the growth of the cell so that the peptidoglycan is not limiting. Teichoic acids are required for the interaction of bacteria with their surroundings, for the regulation of other enzyme activities on the cell surface and contribute to the maintenance of the overall shape of the cell. Capsules are often required to protect pathogens against components of the immune system. Teichoic acids, capsular polysaccharides and peptidoglycan are synthesised in multiple, independent steps by a series of specific biological catalysts, called enzymes. Teichoic acids and capsular polysaccharides are synthesised inside the cell, whereas peptidoglycan is synthesised outside the cell. The lipid-linked teichoic acid precursor is transported across the membrane and transferred onto peptidoglycan, although the precise stage in the synthesis of peptidoglycan that this transfer step occurs is unknown. Only this attachment of the major anionic polymers (wall teichoic acid and capsule) to peptidoglycan builds the final, functional cell wall essential for bacterial lifestyle. We have discovered the enzyme family, called LCP, which performs this transfer step, and in this proposal we seek to understand the molecular basis for its biochemical properties. Specifically, we will study the biochemical reaction catalysed by these enzymes and determine high resolution 3-dimensional structures of the enzymes in complex with the reaction components. We will also develop the synthesis, catalysed by the known enzymes in the pathway, of teichoic acid building blocks that will be better suited to the study of the biochemistry of these proteins than the commercially available components that we have used thus far.