Urease is an essential virulence factor in Proteus spp., contributing to urinary tract infections through the hydrolysis of urea as an energy source and by alkalinizing the environment. Acetohydroxamic acid (AHA) is a reversible urease inhibitor, clinically approved in some countries for the treatment of chronic infections caused by urease-positive bacteria such as Proteus mirabilis.

To investigate the binding mode of AHA to the apo and holo forms of urease from clinical isolates of Proteus spp., evaluating its interactions with catalytic residues, determining whether these residues are conserved among isolates, and assessing the contribution of Ni²⁺ metal coordination.

Structural models were predicted using AlphaFold3, cofactors were added with AlphaFill, and molecular docking was performed with AutoDock Vina. Interactions were analyzed with ChimeraX (v1.10), and residue conservation was verified through multiple sequence alignment.

AHA exhibited predicted affinities of -4.2 kcal/mol (apo) and -4.4 kcal/mol (holo), slightly higher than those of urea (-3.811 and -4.078 kcal/mol). In the apoenzyme, AHA formed hydrogen bonds with His134, His136, His219, His246, Asp360, and Ala363. In the holoenzyme, in addition to maintaining interactions (His219 and Ala363), AHA directly coordinated with both Ni²⁺ ions, an exclusive feature of the holo form of urease. The interactions of AHA with the holo form are strengthened by both Ni²⁺ ions and lysine carboxylation (KCX). The involvement of the modified lysine highlights its functional role not only in metal stabilization but also in ligand anchoring through hydrogen bonding.

Taken together, these findings confirm the inhibitory potential of AHA against Proteus urease and reinforce its clinical relevance as an anti-virulence strategy through competitive inhibition of urea.