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Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2′-phosphotransferase (Tpt1) ‘poison’ that traps the enzyme on its abortive RNA-2′-PO4-(ADP-2″OMe-ribose) reaction intermediate

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2024

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Nucleic Acids Research. Oxford University Press (OUP). 2024, 52(17), S. 10533-10542. ISSN 0305-1048. eISSN 1362-4962. Verfügbar unter: doi: 10.1093/nar/gkae695

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RNA 2′-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2′-PO4 via a two-step mechanism in which: (i) the 2′-PO4 attacks NAD+ C1″ to form an RNA-2′-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2′-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 ‘poison’ that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2′-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2′-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes.

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ISO 690ARNOLD, Jakob, Shreya GHOSH, Renata KASPRZYK, Marcel BRAKONIER, Markus HANNA, Andreas MARX, Stewart SHUMAN, 2024. Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2′-phosphotransferase (Tpt1) ‘poison’ that traps the enzyme on its abortive RNA-2′-PO4-(ADP-2″OMe-ribose) reaction intermediate. In: Nucleic Acids Research. Oxford University Press (OUP). 2024, 52(17), S. 10533-10542. ISSN 0305-1048. eISSN 1362-4962. Verfügbar unter: doi: 10.1093/nar/gkae695
BibTex
@article{Arnold2024-09-23Chemi-70714,
  year={2024},
  doi={10.1093/nar/gkae695},
  title={Chemical synthesis of 2″OMeNAD<sup>+</sup> and its deployment as an RNA 2′-phosphotransferase (Tpt1) ‘poison’ that traps the enzyme on its abortive RNA-2′-PO<sub>4</sub>-(ADP-2″OMe-ribose) reaction intermediate},
  number={17},
  volume={52},
  issn={0305-1048},
  journal={Nucleic Acids Research},
  pages={10533--10542},
  author={Arnold, Jakob and Ghosh, Shreya and Kasprzyk, Renata and Brakonier, Marcel and Hanna, Markus and Marx, Andreas and Shuman, Stewart}
}
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