20.500.12556/RUL-125358 Methylation of selenocysteine catalysed by thiopurine S-methyltransferase 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. binding analysis enzymatic methylation NMR spectroscopy S-adenosylmethionine selenocysteine thiopurine S-methyltransferase jedrska magnetna resonanca analizne metode S-metiltransferaze true false true Angleški jezik Slovenski jezik Članek v reviji 2021-03-12 10:44:39 2021-03-12 10:44:43 2023-03-20 08:53:41 0000-00-00 00:00:00 2019 0 0 Str. 182-190 iss. 1 Vol. 1863 2019 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 543.429.23:615 0304-4165 10.1016/j.bbagen.2018.10.002 4616561 20760839 RAZ_Urbancic_Dunja_2019.pdf RAZ_Urbancic_Dunja_2019.pdf 1 EB748C6696147516FF871D1F39231964 c1cbb2034caa62ce2e2547cc8c6e0621c465f52bcb89a2c94741f22a34c6d6ec b1e50349-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=141046 https://www.sciencedirect.com/science/article/pii/S0304416518303155 1 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=141045 Fakulteta za farmacijo 0 0 0