J Mol Biol. 2025 Jul 31:169368. doi: 10.1016/j.jmb.2025.169368. Online ahead of print.
ABSTRACT
Ureaplasma is one of the smallest pathogenic bacteria, generating approximately 95% of its adenosine triphosphate (ATP) solely through urease. Studies on Ureaplasma parvum, a species of Ureaplasma, have confirmed that adding urease inhibitors inhibits bacterial growth. The Km and Vmax of the urease-mediated reaction were estimated to be 4.3±0.2 mM and 3,333.3±38.0 μmol NH3/min/mg protein, respectively. The cryo-electron microscopy (cryo-EM) structure of Ureaplasma parvum urease (UPU) at a resolution of 2.03 Å reveals a trimer of heterotrimers comprising three proteins: UreA, UreB, and UreC. The active site is well conserved among the known ureases. However, the Vmax of UPU was higher than that of most known ureases, including those ureases derived from Sporosarcina pasteurii (SPU) and Klebsiella aerogenes (KAU) with identical oligomeric state. All-atom molecular dynamics simulations showed that the flap and UreB are more open in UPU than SPU and KAU. His-tagged wild-type recombinant UPU (WT-rUPU) revealed estimated Km and Vmax values of 4.1±0.3 mM and 769.2±7.4 µmol NH3/min/mg protein, respectively. Amino acid substitutions of recombinant UPUs within the flap region to SPU. Amongst the flap region variants, the Vmax of K331N variant was 48-fold lower than that of WT-rUPU. ICP-MS analysis reveals that one molecule of UPU, WT-rUPU, and K331N-rUPU contains 3.7, 0.8, and 0.1 Ni2+ atoms, respectively, suggesting that a wide-open flap of urease may contribute to delivering nickel into the enzyme, resulting in a high Vmax. Ureaplasma evolved highly efficient UPU through a few amino acid substitutions in the disorganized loop of the mobile flap region.
PMID:40752870 | DOI:10.1016/j.jmb.2025.169368
