Investigation of O-acetylserine sulfhydrylase enzymes from bacterial pathogens as potential drug targets

Abstract

Antimicrobial resistance (AMR) has emerged as a formidable global health crisis, necessitating the urgent development of novel antimicrobial strategies. Among the most concerning drug-resistant pathogens are Neisseria gonorrhoeae and Staphylococcus aureus, both of which lack a functional sulfate reduction pathway, rendering them reliant on alternative sulfur acquisition mechanisms. A key enzyme in the de novo biosynthesis of L-cysteine, O-acetylserine sulfhydrylase (CysK), represents a promising target for antimicrobial intervention due to its essential role in sulfur metabolism and bacterial survival. This thesis presents the first comprehensive biochemical and structural characterization of CysK from N. gonorrhoeae (NgCysK) and S. aureus (SaCysK), providing critical insights into their enzymatic function and potential for therapeutic targeting.

In this thesis, we present the structural characterisation of NgCysK and the model used for structure-based virtual inhibitor screening to identify potential NgCysK inhibitors. Virtual screening produced five hit compounds that were tested in vitro, giving compound 5 with inhibition in the mid micromolar range. Analysis of compound 5 docking indicates interactions with the NgCysK active site residues through hydrogen bonds and supporting hydrophobic interactions. This is the first reported inhibitor of NgCysK and provides a promising starting point for developing new antimicrobial adjuvants for treatment of gonorhoea infections.

Utilizing kinetic assays and small-angle X-ray scattering (SAXS), we elucidated the catalytic properties and structural dynamics of NgCysK and SaCysK, demonstrating their distinct substrate specificity. SAXS analysis indicates both enzymes are a single dimeric species in solution with no conformational changes during substrate binding. Combined with gel chromatography analysis, we showed the inability of N. gonorrhoeae to form the cysteine synthase complex by SAXS analysis.

Overall, this research underscores the significance of CysK as a viable antimicrobial target, contributing to the broader efforts of combatting AMR through metabolic pathway inhibition. By integrating structural biology, enzymology, and computational drug discovery, this thesis advances our understanding of bacterial sulfur metabolism and lays the foundation for the rational design of next-generation antimicrobial therapies against drug-resistant N. gonorrhoeae and S. aureus.