| dc.description.abstract | Coxiella burnetii, the causative agent of Q fever, is a versatile, highly infectious select agent. Due to the immunogenic nature of its LPS-linked O-antigen, and this conveying the main determinant of virulence, a synthetic biology approach is being applied to study this molecule, with a view towards glycoconjugate vaccine design. This thesis presents a dual approach to study of the C. burnetii O-antigen: direct characterisation of O-antigen biosynthetic enzymes, and the design of a phenol-free method by which to extract LPS material. For the former, studies focussed on characterisation of CBU_1838 as a dTDP-sugar isomerase proposed to be involved in the biosynthesis of DHHS, an O-antigen component, and a sugar unique to Coxiella. Characterisation through spectrophotometric assays, analysis of solvent exchange, and structural studies were run in parallel with another uncharacterised dTDP-sugar isomerase, StrM from Streptomyces griseus. From both kinetic assays coupled to the E. coli rhamnose-biosynthetic enzymes, and studies of solvent exchange, it is shown here that both of these query dTDP-sugar isomerases catalyse double-epimerisation of dTDP-4-keto-6-deoxy-glucose at positions 3″ and 5″. Additionally, structural studies of CBU_1838 revealed a single amino acid difference in the active site, compared to a panel of reference dTDP-sugar isomerases. From analysis of dTDP binding, and the docking of substrate-analogs, it is clear that this single residue could allow stabilisation of the hypothesised true substrate of CBU_1838, dTDP-4-keto-6-hydroxy-glucose. Surreptitiously, preparative experiments for studies of dTDP-sugar isomerase-catalysed solvent exchange led to a novel discovery about the dTDP-glucose 4,6-dehydratase, RmlB: whilst the overall reaction is irreversible, enzyme-mediated solvent exchange at position C5″ occurs when EcRmlB is incubated with its product, dTDP-4-keto-6-deoxy-glucose. The second approach taken to O-antigen studies, a novel method for LPS extraction, unfortunately did not yield tangible results. However, methods for growth of the Nine Mile II strain of C. burnetii were optimised, and areas for extraction improvement have been highlighted. | en_GB |
