Structural and Biochemical Characterisation of Ribitol-5-phosphate Transferases

Wall teichoic acids (WTAs) play a role in cell growth, division and morphology as well as colonisation and adhesion in Gram-positive bacterial species such as Staphylococcus aureus and Bacillus subtilis. In S. aureus and B. subtilis W23, WTAs largely consists of a ribitol-5-phosphate (RboP) polymer, which is synthesised by TarK and TarL enzymes using CDP-ribitol (CDP-Rbo). This donor substrate, CDP-Rbo, is synthesised from RboP by TarI. Deletion of the enzymes along the WTA pathway results in restored antibiotic susceptibility in methicillin-resistant S. aureus, demonstrating the potential of WTA biosynthesis as a therapeutic target. Therefore the characterisation of the Tar enzymes could aid in the development of novel antimicrobial agents. This project endeavoured to facilitate the generation of the difficult-to-synthesise donor substrate, CDP-Rbo, via TarI and to characterise the TarK and TarL enzymes, thereby providing insight into the protein activity and 3D structures.
Firstly, CDP-Rbo synthesis was achieved using a chemoenzymatic approach utilising TarI with RboP produced by members of the group via chemical synthesis. Efforts were then made to recombinantly express a range of TarK/L homologues which highlighted that large set of expression trials were required to produce soluble protein. Recombinant TarK and TarL from B. subtilis W23 were successfully solubilised by using Rosetta2 plysS E. coli and, for TarL, changing the His6 tag location to the C-terminal end. Buffers containing CHAPS were required, therefore aiding in solubility throughout the multi-step chromatography protocols. Once the target proteins and variants of interest were purified, stability was assessed using circular dichroism and nanoDSF with/without ligands. TarK activity was measured by liquid chromatography–mass spectrometry and could utilise CDP-Rbo. Crystallisation was not successful, but mass photometry results identified multimeric forms of TarL ideal for cryo-EM. 2D classes then yielded a possible tetrameric form of TarL facilitating further structural studies using this technique.