Cysteine biosynthesis and the role of CysE in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is the causative organism of the sexually transmitted infection (STI), gonorrhoea. Over decades of antibiotic use, the emergence of antibiotic-resistant strains of N. gonorrhoeae has dramatically increased and we are facing the reality of almost untreatable gonorrhoea. Combined with increasing incidence, there is an urgent need for new antimicrobial treatments for gonorrhoea infection. The synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays an important role in protein molecules, but also in the synthesis of glutathione for protection against oxidative stress. In addition, cysteine is the key step for the incorporation of sulphur into a variety of cellular constituents. N. gonorrhoeae displays unique differences in assimilation of sulphate for the synthesis of cysteine, yet little is known about the synthesis of this important amino acid in N. gonorrhoeae.

The first step in the dual-step cysteine biosynthesis pathway is catalysed by the serine acetyltransferase, CysE, an essential gene in N. gonorrhoeae. To design inhibitors against this key enzyme we need to elucidate the enzymatic mechanism and the three-dimensional structure. In this thesis, we determine the kinetic parameters and regulation of CysE and present the structure of CysE to 2.01 Å. Biochemical assays demonstrate that CysE has serine acetyltransferase activity and experiences substrate inhibition by acetyl CoA. CysE is also sensitive to feedback inhibition by L-cysteine, which competitively inhibits CysE relative to the substrate L-serine. The structure shows CysE belongs to the left-handed β helix family and adopts a hexameric structure consisting of a dimer of trimers. Our structure reveals the first evidence of domain swapping for a CysE structure, where C-terminal tails domain swap with neighbouring monomers in the crystal lattice. Although an artefact of crystallisation it provides insight into the flexibility of this region.

Collectively the data presented in this thesis represents key advances in our understanding of the uncharacterised cysteine biosynthetic pathway in N. gonorrhoeae. Data presented here is the basis for future work using the structure of CysE to guide computational inhibitor design, to identify lead compounds for CysE inhibition, and for the development of new antimicrobials for treatment of gonorrhoea.