Urease is a nickel-dependent enzyme linked to the pathogenesis of several bacterial species[1], including carbapenemase-producing Klebsiella, which are often associated with hospital infections and multidrug resistance to antimicrobials. N-acetylcysteine (NAC), a clinically approved compound with mucolytic properties, has also shown in vitro inhibitory activity against Klebsiella pneumoniae urease[2] and other bacteria[3].

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

Sequences were aligned, and structural models were generated with AlphaFold3 (apoenzyme) and AlphaFill (holoenzyme), with insertion of Ni²⁺ ions. Docking was performed with AutoDock Vina, and interactions were evaluated using the Protein–Ligand Interaction Profiler (PLIP). The obtained affinities were compared with those of urea as the reference natural substrate.

Docking simulations showed strong binding for both forms: -4.8 kcal/mol for the apoenzyme and -5.2 kcal/mol for the holoenzyme. In both states, NAC interacted with a conserved set of residues. Shared interactions included hydrogen bonds with His219 and Asp360, and salt bridges with His134, His136, His219, and His272. In the apoenzyme, a specific hydrogen bond with His246 was observed, whereas in the holoenzyme, NAC formed additional hydrogen bonds with Ala363 and metal coordination with Ni²⁺ ions, absent in the apo form. Despite these subtle differences, the interaction core was highly conserved. Multiple sequence alignment of 438 urease sequences from Klebsiella clinical isolates confirmed that all interacting residues are highly conserved across the genus, indicating functional and structural robustness of the NAC binding interface.

These results indicate that NAC has the potential to interact with conserved urease residues, although full inhibition may depend on enzymatic maturation and the presence of metal ions. This binding potential supports the repositioning of NAC as an anti-virulence compound against infections caused by multidrug-resistant Klebsiella spp.