Distinct contributions of O-acetylserine sulfhydrylases to cysteine biosynthesis in Pseudomonas aeruginosa.

Cysteine biosynthesis in bacteria proceeds primarily via the de novo pathway, involving serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (OASS). This pathway is absent in humans, and its inhibition impairs microbial fitness, virulence, and antibiotic resistance, making its enzymes attractive antimicrobial targets. Most bacteria encode two OASS isoforms: CysK, which forms the cysteine synthase complex (CSC) with CysE, and CysM, which typically acts independently. While conserved, their biochemical properties and regulatory roles vary across species. Here, we investigated CysK (PaCysK) and CysM (PaCysM) from Pseudomonas aeruginosa, an opportunistic pathogen of major concern due to intrinsic antibiotic resistance. We characterized their steady-state and pre-steady-state kinetics, structural features, and assessed substrate preferences through microbiological analyses of cysK and cysM deletion mutants. Our results revealed that PaCysK and PaCysM play redundant yet critical roles in cysteine biosynthesis in P. aeruginosa. PaCysK exhibits optimal activity with sulfide, supporting its primary function in sulfide-dependent cysteine biosynthesis. In contrast, PaCysM shows broader specificity, catalyzing cysteine formation from both sulfide and thiosulfate, suggesting a specialization for alternative sulfur sources. Structural modeling supports this, revealing PaCysM active-site features that facilitate thiosulfate binding and turnover. We also tested PaCysK binding to a synthetic peptide mimicking the C-terminal region of P. aeruginosa CysE1 and found no interaction, suggesting that the CSC does not form in P. aeruginosa. In contrast, PaCysK binds the equivalent peptide from Salmonella enterica serovar Typhimurium CysE, known to mediate CSC formation, confirming that the enzyme retains the capacity for protein-protein interactions through a canonical CSC-like mechanism.