N-acetylcysteine (NAC), a mucolytic widely used in medical practice for the treatment of viral infections, has also shown antimicrobial properties through the inhibition of the bacterial urease of Proteus mirabilis [1]. Urease is a nickel-dependent enzyme that hydrolyzes urea into ammonia and carbon dioxide, promoting bacterial persistence through an increase in local pH. In Acinetobacter spp., a genus frequently associated with multidrug-resistant infections, urease is a virulence factor critical for persistence in macrophages during lung infection [2].

This study evaluated the binding of NAC to the immature (apoenzyme) and mature (holoenzyme) forms of urease from clinical Acinetobacter isolates.

The structures were modeled using AlphaFold3 (apo) and AlphaFill (holo), the latter including Ni²⁺ cofactors in the active site. NAC was submitted to molecular docking with AutoDock Vina (v1.2.7) in both forms. The interactions were analyzed in PoseView, and the conservation of residues was verified by multiple alignment of 233 sequences of clinical isolates using MAFFT (v7.505).

Docking simulations suggested similar binding affinities for the apoenzyme and holoenzyme, –4.7 kcal/mol and –4.4 kcal/mol, respectively. NAC interacted with an overlapping set of residues in both forms, including His133, His135, His218, His245, His271, and Ala362, mainly through hydrogen bonds and salt bridges. In the apoenzyme, a salt bridge was observed with Lys216, while in the holoenzyme, NAC formed a specific hydrogen bond with Asp359 and coordinated both Ni²⁺ ions — an interaction absent in the apo form. These subtle differences reflect the structural changes associated with enzymatic maturation, but overall, the binding mode was highly conserved. The multiple alignment of 233 Acinetobacter spp. urease sequences from clinical isolates confirmed that all interacting residues are highly conserved, reinforcing their functional importance.

The ability of NAC to bind to structurally essential residues of the active site of urease from clinical Acinetobacter spp. suggests the potential repositioning of this drug for the attenuation of virulence in clinical infections by multidrug-resistant Acinetobacter baumannii.