Abstract
2′-Deoxyuridine 5′-triphosphate nucleotide hhydrolase (Dut) hydrolyzes dUTP to dUMP and pyrophosphate to prevent erroneous incorporation of dUMP from the dUTP metabolic pool into DNA. Dut is considered as a promising pharmacological target for antimetabolite therapy. Enzymatically active Dut is a trimer that binds the substrate at the interface between the subunits. High-speed nanoscale differential scanning fluorimetry (nanoDSF) was used to study how various physicochemical factors affect the stability of the Escherichia coli Dut trimer. Unlike with monomeric proteins, thermal unfolding of Dut occurred in two steps, the first one corresponding to dissociation of the trimer into monomeric subunits. Hydrophobic interactions and hydrogen bonds at the interfaces between the subunits were found to contribute most to trimer stabilization. The binding of nucleotide ligands partly stabilized the Dut trimer. In general, nanoDSF is a convenient assay for screening low-molecular-weight compounds for their ability to destabilize the active Dut trimer.
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ACKNOWLEDGMENTS
DNA sequencing was performed at the Siberian Branch of the Russian Academy of Sciences Genomics Core Facility.
Funding
This work was supported by the Strategic academic leadership program of Tomsk State University (Priority-2030). Structural analysis was supported by the Ministry of Science and Higher Education of the Russian Federation (agreement no. 075-15-2022-263).
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A. V. Yudkina and E. A. Kovalenko contributed equally to this work.
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The authors declare that they have no conflicts of interest. This article does not contain any studies involving animals or human subjects performed by any of the authors.
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Translated by T. Tkacheva
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Yudkina, A.V., Kovalenko, E.A., Endutkin, A.V. et al. Factors Affecting the Stability of the Trimer of 2'-Deoxyuridine 5'-Triphosphate Nucleotide Hydrolase from Escherichia coli. Mol Biol 57, 312–319 (2023). https://doi.org/10.1134/S002689332302022X
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DOI: https://doi.org/10.1134/S002689332302022X