Characterisation of O-acetylserine sulfhyrdrylase (CysK) enzymes from bacteria lacking a sulfate reduction pathway

Sulfur metabolism plays an important role in bacterial pathogenesis. Elucidation of differences in sulfur metabolism across bacterial pathogens furthers our understanding of host survival and offers opportunities to disrupt these pathways for new therapies. Withing bacteria sulfur metabolism converges at the synthesis of L-cysteine. One of the key mechanisms of obtaining sulfur for the synthesis of L-cysteine is the successive reduction of sulfate to sulfide via the sulfate reduction pathway. Accordingly, L-cysteine biosynthesis is a critical metabolic pathway for bacterial survival, particularly in pathogenic species such as Neisseria gonorrhoeae and Staphylococcus aureus , which lack the sulfate reduction pathway. O-acetylserine sulfhydrylase catalyses the second step of the two-step synthesis reaction, condensing sulfide or thiosulfate (in the case of OASS-A/CysK or OASS-B/CysM respectively) with O-acetylserine to synthesize cysteine. Here we investigate the enzymatic properties and functional characterization of O-acetylserine sulfhydrylase, from N. gonorrhoeae and S. aureus , with a focus on substrate specificity, kinetic parameters, and cysteine synthase complex (CSC) formation. Using small angle X-ray scattering and kinetic assays we demonstrate that both N. gonorrhoeae and S. aureus CysK enzymes utilise only sodium sulfide for the synthesis of cysteine, despite the lack of a sulfate reduction pathway (to generate sulfide) in these organisms. Both enzymes demonstrate a higher affinity for O -acetylserine (OAS) compared to sodium sulfide (Na ₂ S). We also show that the two cysteine synthesis enzymes, CysE and CysK that traditionally form the cysteine synthase complex do not form a complex in N. gonorrhoeae . These findings highlight the functional divergence in sulfur metabolism strategies among bacteria lacking sulfate reduction and provide deeper insights into the adaptive mechanisms of N. gonorrhoeae and S. aureus in sulfur flux.