RESUMO
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+ C1â³ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2â³ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1â³,2â³-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 2â³OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2â³OMeNAD+ 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-2â³OMe-ribose) intermediate. Although 2â³OMeNAD+ 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 2â³-substitutions may prove advantageous in targeting the fungal enzymes.