Structure function analysis of O-antigen modifying enzymes in the bacterial pathogen Salmonella

Carbohydrates are essential for bacterial survival. As well as being taken up and used as a carbon source, they are synthesised by bacteria to protect them from environmental stresses and assist in colonisation of new environments. Bacteria possess a range of mechanisms to modify the diverse carbohydrate structures that they synthesise, a key example being O acylation. Acylation of bacterial carbohydrates can have applications in processes such as antigenic variation, osmoregulation, virulence and cell division, and can also have transferrable applications in clinical and industrial processes.
The aim of this research was to investigate the mechanism of carbohydrate O-acylation by Acyltransferase_3 (AT3) domain containing proteins OafA and OafB of the bacterial pathogen Salmonella. These proteins contain a membrane bound AT3 domain fused to an extra cytoplasmic SGNH domain. They O-acetylate different residues in the variable repeating carbohydrate O-antigen of lipopolysaccharide.
The research presented in this thesis demonstrates that both the AT3 and SGNH domains are required for O-antigen acetylation in these proteins. It also highlights functional residues that support a conserved mechanism of transmembrane acyl group transport by AT3 domain-containing proteins, and suggests residues within the AT3 domain specifically adapted for function with a fused SGHN domain. Furthermore, structural and functional characterisation of the SGNH domain of OafA and OafB supports the hypothesis that this domain is responsible for the final step of acetyl group transport to the carbohydrate acceptor in this system, and also identifies how acceptor substrate specificity is achieved by this domain.
These findings have allowed a refined mechanistic model for AT3-SGNH fused proteins to be defined, enhancing our understanding of an important family of acyltransferase proteins which are found across all domains of life. This work therefore provides a framework for understanding and potentially manipulating these proteins, to enable carbohydrates of clinical and industrial significance to be engineered.