Chemical synthesis of 2OMeNAD(+) and its deployment as an RNA 2 -phosphotransferase (Tpt1) ‘poison’ that traps the enzyme on its abortive RNA-2 -PO(4)-(ADP-2OMe-ribose) reaction intermediate Journal Article
| Authors: | Arnold, J.; Ghosh, S.; Kasprzyk, R.; Brakonier, M.; Hanna, M.; Marx, A.; Shuman, S. |
| Article Title: | Chemical synthesis of 2OMeNAD(+) and its deployment as an RNA 2 -phosphotransferase (Tpt1) ‘poison’ that traps the enzyme on its abortive RNA-2 -PO(4)-(ADP-2OMe-ribose) reaction intermediate |
| Abstract: | 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. © The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research. |
| Keywords: | rna; adenosine diphosphate ribose |
| Journal Title: | Nucleic Acids Research |
| Volume: | 52 |
| Issue: | 17 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2024-09-23 |
| Start Page: | 10533 |
| End Page: | 10542 |
| Language: | English |
| DOI: | 10.1093/nar/gkae695 |
| PUBMED: | 39162230 |
| PROVIDER: | scopus |
| PMCID: | 39162230 |
| DOI/URL: | |
| Notes: | Article -- Source: Scopus |
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