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 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.