Structural Analysis and Molecular Dynamics Simulations of Urease From Ureaplasma parvum.

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 K_m and V_max 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 V_max 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 K_m and V_max 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 V_max 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 V_max. Ureaplasma evolved highly efficient UPU through a few amino acid substitutions in the disorganized loop of the mobile flap region.