The genus Pseudomonas includes opportunistic pathogens frequently exhibiting multidrug resistance and involved in hospital infections, in which urease remains an unexplored therapeutic target for virulence attenuation. Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide, contributing to bacterial adaptation through environmental pH increase. Acetohydroxamic acid (AHA) is a well-characterized reversible urease inhibitor, approved in some countries for treating chronic urinary tract infections.

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

Structural models were generated using AlphaFold3 (apoenzyme) and modified with AlphaFill (holoenzyme) for nickel ion insertion. Docking was performed using AutoDock Vina, and interactions were analyzed structurally in ChimeraX (v1.10). Interactions with AHA and with urease’s natural substrate, urea, were analyzed.

Docking simulations revealed similar binding affinities for AHA in both forms, –4.3 kcal/mol (apoenzyme) and –4.4 kcal/mol (holoenzyme); and for urea, –3.9 and –4.1 kcal/mol, respectively. In both cases, AHA interacted with a conserved set of catalytic residues, including His218, His245, His359, and Ala362. In the apoenzyme, AHA also interacted via hydrogen bonds with His133 and His135, while in the holoenzyme it established additional interactions with His271, KCX (carboxylated lysine), and coordinated with both nickel ions. Although AHA interacted with catalytic residues in both forms, stronger interactions were observed through metal coordination with Ni in the holoenzyme.

These findings reinforce the potential of AHA as a broad-spectrum urease inhibitor and highlight the need for further studies on its use in infections caused by Pseudomonas, beyond current therapeutic indications for urinary and gastric infections caused by Proteus mirabilis and Helicobacter pylori.