Phosphotransferases in bacterial cell wall biosynthesis

细胞壁是细菌的两个主要分支之一——革兰氏阳性菌——的一个重要的、有时被忽视的关键方面。没有细胞壁，革兰氏阳性细菌细胞将受到严重损害，细胞的可溶性内容物将冲破环绕细胞的相当脆弱的单一脂质膜。细胞会死亡。这些生物的细胞壁由两个主要组成部分组成，一个是交联肽聚糖链的网状结构，它附着在长而聚合的碳水化合物上，如磷壁酸和胶囊。肽聚糖和壁壁酸都起着关键作用——肽聚糖提供了必要的物理保护，免受细胞渗透压的影响，它的合成与细胞的生长相协调，因此肽聚糖不受限制。天藻酸是细菌与周围环境相互作用所必需的，是调节细胞表面其他酶活性所必需的，也是维持细胞整体形状所必需的。通常需要胶囊来保护病原体免受免疫系统成分的侵害。天衣酸、荚膜多糖和肽聚糖是通过一系列被称为酶的特定生物催化剂在多个独立步骤中合成的。天衣酸和荚膜多糖在细胞内合成，而肽聚糖则在细胞外合成。脂质连接的壁壁酸前体通过细胞膜转移到肽聚糖上，尽管这种转移发生在肽聚糖合成的确切阶段尚不清楚。只有这种主要阴离子聚合物（壁苔酸和胶囊）附着在肽聚糖上，才能构建最终的、功能性的细胞壁，这是细菌生活方式所必需的。我们已经发现了一种叫做LCP的酶家族，它执行了这一转移步骤，在这项提议中，我们试图了解其生化特性的分子基础。具体而言，我们将研究这些酶催化的生化反应，并确定酶与反应组分配合物的高分辨率三维结构。我们还将开发合成方法，在已知的酶的催化下，合成磷壁酸构建块，这将比我们迄今为止使用的商业上可用的成分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.