N-acetylcysteine (NAC), a mucolytic agent widely used in viral respiratory infections, also inhibits biofilm formation by Pseudomonas aeruginosa [1]. In addition to biofilm, urease, a nickel-dependent metalloenzyme, is a virulence factor that contributes to bacterial pathogenesis by hydrolyzing urea into ammonia and carbon dioxide, raising the local or intraphagosomal pH and resisting phagocytosis [2]. In Pseudomonas spp., opportunistic pathogens frequently associated with hospital infections, urease represents a therapeutic target to be explored for attenuating the virulence of multidrug-resistant strains.

In this study, we investigated the binding of NAC to the immature (apoenzyme) and mature (holoenzyme) forms of Pseudomonas spp. urease.

Structural models were generated using AlphaFold3 (apoenzyme) and AlphaFill (holoenzyme), incorporating nickel ions into the holoenzyme active site. Interactions were analyzed with PyMOL (v4.3), and residue conservation was verified by aligning 216 clinical ureases with MAFFT.

Docking simulations of NAC revealed strong affinities: –4.9 kcal/mol with the apoenzyme and –5.1 kcal/mol with the holoenzyme, whereas the affinity for urea was only –4.0 kcal/mol for both forms. NAC bound to a largely overlapping and functionally relevant set of residues in both forms. In the apoenzyme, it formed hydrogen bonds with His218, His245, and His271, and established salt bridges with His133, His135, Lys216, His218, His245, and His271. In the holoenzyme, NAC formed hydrogen bonds with Ala166, Arg335, Asp359, and Ala362; salt bridges with His133, His135, His218, and His271; and also directly interacted with Ni²⁺ ions. To evaluate the conservation of these residues, we performed multiple sequence alignment of 216 ureases from clinical Pseudomonas isolates. All residues involved in interactions with NAC, such as hydrogen bonds, salt bridges, and metal coordination, were highly conserved, supporting their essential roles in enzymatic function and conservation in ligand recognition across different isolates.

NAC has the potential to interact with conserved urease residues in clinical isolates of Pseudomonas spp.; however, full inhibition may depend on enzyme maturation and the presence of metal ions, for its repositioning as a compound for virulence attenuation.