Substrate analogs that trap the 2'-phospho-ADP-ribosylated RNA intermediate of the Tpt1 (tRNA 2'-phosphotransferase) reaction pathway

The enzyme Tpt1 removes an internal RNA 2'-PO4 via a two-step reaction in which: (i) the 2'-PO4 attacks NAD(+) 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. Because step 2 is much faster than step 1, the ADP-ribosylated RNA intermediate is virtually undetectable under normal circumstances. Here, by testing chemically modified nucleic acid substrates for activity with bacterial Tpt1 enzymes, we find that replacement of the ribose-2'-PO4 nucleotide with arabinose-2'-PO4 selectively slows step 2 of the reaction pathway and results in the transient accumulation of high levels of the reaction intermediate. We report that replacing the NMN ribose of NAD(+) with 2'-fluoroarabinose (thereby eliminating the ribose O2'' nucleophile) results in durable trapping of RNA-2'-phospho-(ADP-fluoroarabinose) as a "dead-end" product of step 1. Tpt1 enzymes from diverse taxa differ in their capacity to use ara-2''F-NAD(+) as a substrate.