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

Abstract

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 RNA 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.