Phosphotransferases in bacterial cell wall biosynthesis

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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.