Structural and biochemical characterisation of O-acetylserine sulphydrylase (CysK) from Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is the causative bacteria of the sexually transmitted infection (STI), gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to almost untreatable gonorrhoea over the last few decades. There is an urgent need for development of new antimicrobial treatments for gonorrhoea infection. Synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays a vital role in protein folding and function, and synthesis of glutathione for protection against oxidative stress during infection. The final step of the two-step cysteine biosynthesis pathway is catalysed by the enzyme O-acetylserine sulphydrylase (OASS). Most bacteria have two isoforms of OASS, OASS-A/CysK that utilises sulphide and O-acetylserine for the synthesis of cysteine and OASS-B/CysM that utilises thiosulphate. N. gonorrhoeae has just one OASS isoform (annotated as CysK) in its genome. N. gonorrhoeae displays unique differences in its sulphate acquisition pathway for cysteine biosynthesis, yet little is known about these pathways in N. gonorrhoeae. We propose CysK is a potential antimicrobial target. This thesis biochemically characterises the structure and function of CysK to determine its role in cysteine biosynthesis. Kinetic characterisation demonstrates that CysK has O-acetylserine sulphydrylase activity and displays positive cooperativity with respect to substrates, O-acetylserine and sulphide. Sulphide shows partial allosteric inhibition, and the enzyme does not use thiosulphate as a substrate. The structure of CysK was solved to 2.49 Å by X-ray crystallography and shows CysK belongs to the tryptophan synthase β superfamily and adopts a homodimeric structure consisting of two monomers. The structure supports positive cooperativity as co-factor binding residues are in inactive and active conformations in each monomer of the dimer, respectively. Many CysK enzymes form complexes with the first enzyme in the two-step cysteine synthesis pathway, CysE. It appears CysE and CysK from N. gonorrhoeae are unable to form a complex, indicating an alternate pathway for regulation of sulphur flux and cysteine production. Attempts to investigate the in vivo role of CysK were inconclusive. The research presented in this thesis represents a major leap in our understanding of the uncharacterised cysteine biosynthesis pathway in N. gonorrhoeae. Data presented here is the basis for future work using the kinetics and structure of CysK to guide computational inhibitor design, to identify lead compounds for CysK inhibition, and thereby development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.