Factors Affecting the Stability of the Trimer of 2'-deoxyuridine-5'-triphosphate Nucleotide Hydrolase from  Escherichia coli

A. V. Yudkina; Юдкина А. В.; E. A. Kovalenko; Коваленко Е. А.; A. V. Endutkin; Ендуткин А. В.; E. P. Panferova; Панфёрова Е. П.; A. A. Kirilenko; Кириленко А. А.; A. A. Kokhanenko; Коханенко А. А.; D. O. Zharkov; Жарков Д. О.

doi:10.31857/S0026898423020246

Factors Affecting the Stability of the Trimer of 2'-deoxyuridine-5'-triphosphate Nucleotide Hydrolase from Escherichia coli

作者: Yudkina A.V.¹^,2, Kovalenko E.A.³, Endutkin A.V.¹, Panferova E.P.¹, Kirilenko A.A.³, Kokhanenko .A.³, Zharkov D.O.¹^,2
隶属关系:
1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
2. Novosibirsk State University
3. Tomsk State University
期: 卷 57, 编号 2 (2023)
页面: 330-339
栏目: СТРУКТУРНО-ФУНКЦИОНАЛЬНЫЙ АНАЛИЗ БИОПОЛИМЕРОВ И ИХ КОМПЛЕКСОВ
URL: https://journals.rcsi.science/0026-8984/article/view/138655
DOI: https://doi.org/10.31857/S0026898423020246
EDN: https://elibrary.ru/EEJRNT
ID: 138655

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详细

To prevent erroneous incorporation of dUMP into DNA from the dUTP metabolic pool, all living cells contain 2′-deoxyuridine-5′-triphosphate nucleotide hydrolase (Dut), an enzyme that hydrolyzes dUTP to dUMP and pyrophosphate. Dut is considered a promising pharmacological target for antimetabolite therapy. Enzymatically active Dut is a trimer that binds the substrate at the interface between the subunits. Here we use high-speed nanoscale differential scanning fluorometry (nanoDSF) to study how various physicochemical factors affect the stability of the E. coli Dut trimer. Unlike for monomeric proteins, thermal denaturation of Dut occurred in two stages, the first of which corresponds to the dissociation of the trimer to monomeric subunits. Hydrophobic interactions and hydrogen bonds at the interfaces between subunits contributed most to trimer stabilization. The Dut trimer was partially stabilized upon binding of nucleotide ligands. In general, nanoDSF is a convenient assay for screening low molecular weight compounds for their ability to destabilize the active Dut trimer.

关键词

dUTPase, protein–protein interactions, oligomerization, differential scanning fluorimetry

参考

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