Dunja Urbančič (Author), Anita Kotar (Author), Alenka Šmid (Author), Marko Jukič (Author), Stanislav Gobec (Author), Lars Martensson (Author), Janez Plavec (Author), Irena Mlinarič-Raščan (Author)

Abstract

Background Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown. Methods To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and 77Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations. Results Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by 77Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy. Conclusions The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner. General Significance. Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid.

Keywords

Binding analysis;enzymatic methylation;NMR spectroscopy;S-adenosylmethionine;selenocysteine thiopurine;S-methyltransferase;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Organization: UL FFA - Faculty of Pharmacy
Publisher: Elsevier
UDC: 543.429.23:615
COBISS: 4616561 Link will open in a new window
ISSN: 0304-4165
Views: 407
Downloads: 227
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Other data

Secondary language: Slovenian
Secondary keywords: Jedrska magnetna resonanca;
Type (COBISS): Article
Pages: str. 182-190
Volume: ǂVol. ǂ1863
Issue: ǂiss. ǂ1
Chronology: Jan. 2019
DOI: 10.1016/j.bbagen.2018.10.002
ID: 12639649