Klebsiella is a clinically relevant bacterial genus associated with multidrug resistance. Urease is a virulence factor and a potential target for inhibitor-based interventions (Shafik et al., 2023). Acetohydroxamic acid (AHA), a reversible urease inhibitor, has been used clinically to treat infections caused by urease-positive bacteria such as Proteus mirabilis.

To evaluate the binding of AHA to the immature (apoenzyme) and mature (holoenzyme) forms of urease from clinical isolates of Klebsiella spp.

Structural models were generated using AlphaFold3 (apo) and AlphaFill (holo), the latter incorporating Ni²⁺ cofactors in the active site.

Docking simulations revealed binding affinities of –4.2 kcal/mol for the apo form and –4.4 kcal/mol for the holo form. In both forms, AHA interacted with overlapping residue sets, including His219 and Ala363. The apoenzyme formed hydrogen bonds with His134, His136, His219, His246, Asp360, and Ala363. In the holoenzyme, AHA maintained interactions with His219 and Ala363 and additionally formed bonds with Ala167 and both nickel ions, highlighting the structural role of metal coordination in the mature form. Despite these differences, most interacting residues were shared between apo and holo, indicating that AHA targets a structurally conserved binding region. To assess conservation among clinical isolates, multiple sequence alignment was performed on 438 urease sequences from Klebsiella clinical strains. All residues involved in AHA interactions were highly conserved, reinforcing their functional importance and supporting the broad applicability of AHA-based inhibition within this genus. The binding affinities of AHA to the active site were similar to those for the natural substrate urea (–4.0 kcal/mol) and to redocking values obtained with the crystallized structure (1FWE, –4.3 kcal/mol), whose inhibition of the holoenzyme has been experimentally validated in vitro.

These findings demonstrate that AHA can bind to both maturation states of urease, involving conserved catalytic residues, with holo-specific enhancement through metal coordination.