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Published on: April 4, 2014
Structural Analysis and Molecular Dynamics Simulations of Urease From Ureaplasma parvum
Heng Ning Wu1, Junso Fujita2, Yukiko Nakura1
1Department of Developmental Medicine, Research Institute, Osaka Women's and Children's Hospital, Izumi City 594-1101 Osaka, Japan.
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.
Insights
Ureaplasma parvum urease (UPU) is highly efficient due to its open flap structure, which facilitates nickel delivery and high activity. Key amino acid changes in the flap region significantly reduce UPU
Area of Science:
- Biochemistry
- Microbiology
- Structural Biology
Background:
- Ureaplasma is a small pathogenic bacterium relying on urease for energy.
- Urease inhibitors are known to impede Ureaplasma growth.
- The kinetic parameters (Km and Vmax) of Ureaplasma parvum urease (UPU) have been characterized.
Purpose of the Study:
- To elucidate the structural and functional basis for the high catalytic efficiency of Ureaplasma parvum urease (UPU).
- To investigate the role of the flap region and nickel content in UPU's enzymatic activity.
Main Methods:
- Cryo-electron microscopy (cryo-EM) for structural determination of UPU.
- All-atom molecular dynamics simulations to compare UPU with other ureases.
- Site-directed mutagenesis of UPU flap region variants.
- Inductively coupled plasma mass spectrometry (ICP-MS) for nickel quantification.
Main Results:
- The cryo-EM structure of UPU revealed a unique trimer of heterotrimers with an open flap conformation.
- UPU exhibited a significantly higher Vmax compared to ureases from Sporosarcina pasteurii (SPU) and Klebsiella aerogenes (KAU).
- A specific mutation (K331N) in the flap region drastically reduced Vmax and nickel content, while the wild-type UPU had higher nickel content than recombinant variants.
Conclusions:
- The open flap structure of UPU is crucial for its high catalytic efficiency, likely by facilitating nickel cofactor incorporation.
- Specific amino acid substitutions in the flap region are responsible for Ureaplasma's evolution of a highly active urease.
- Understanding UPU's mechanism provides insights into urease function and potential therapeutic targets.
