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 ′ ′ ′ ′ ′ -PO 4 via a two-step reaction in which: (i) the 2 ′ ′ ′ ′ ′ -PO 4 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 ′ ′ ′ ′ ′ -PO 4 nucleotide with arabinose-2 ′ ′ ′ ′ ′ -PO 4 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.

Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential antifungal targets

“…5 C) could, in principle, be able to support the first step in the Tpt1 pathway, but would be unable to undergo transesterification in the second step for lack of an O2″ nucleophile. Previously, we surveyed Tpt1 enzymes from Runella slithyformis , Clostridium thermocellum , Chaetomium thermophilum , and Homo sapiens at 0.5 µM concentration for activity in the presence of 0.2 µM 5′ 32 P-labeled 6-mer 2′-PO 4 -branched RNA oligonucleotide and either 50 µM NAD + or ara-2″F-NAD + ( Dantuluri et al 2020 ). When provided with 50 µM ara-2′′F-NAD + , the Runella and Clostridium enzymes converted nearly all of the substrate into an RNA-2′-phospho-(ADP-fluoroarabinose) dead-end product of step 1 of the Tpt1 pathway.…”

“…In contrast, human Tpt1 effected no detectable reaction of the 2′-PO 4 RNA substrate in the presence of 50 µM ara-2″F-NAD + . We surmised that Tpt1 enzymes from different sources may vary in their sensitivity to the arabinose sugar modification of NAD + , but the utilization of ara-2″F-NAD + as a substrate by Tpt1 does indeed result in trapping of the step 1 reaction product ( Dantuluri et al 2020 ).…”

“…To query the RNA requirement for Tpt1 activity, we exploited a 5′ 32 P-labeled analog of the 6-mer 2′-PO 4 oligonucleotide substrate ( Dantuluri et al 2020 ) in which the five nucleotides flanking the 2′-PO 4 ribonucleotide were replaced by deoxynucleotides ( Fig. 6 ).…”

“…7 E) as substrate for the Runella and Clostridium Tpt1 enzymes. Replacement of the ribose-2′-PO 4 nucleotide with arabinose-2′-PO 4 selectively slowed step 2 of the reaction pathway and resulted in the transient accumulation of high levels of the RNA-2′-arabino-phospho-(ADP-ribose) reaction intermediate ( Dantuluri et al 2020 ). Here we tested activity of the four fungal pathogen Tpt1s with the RNA ara-2′-PO 4 substrate.…”

“…A detailed rationale for this effect is elusive in the absence of a structure of Tpt1 in a binary complex with NAD + . Replacing the NMN ribose of NAD + with 2′-fluoroarabinose can trap RNA-2′-phospho-(ADP-fluoroarabinose) as a dead-end step 1 product in the case of the Runella and Clostridium Tpt1 enzymes, but human Tpt1 was unreactive with ara-2″F-NAD + ( Dantuluri et al 2020 ). Here we found that the four fungal Tpt1 enzymes were feeble in their use of ara-2″F-NAD + as a substrate for step 1 of the Tpt1 pathway.…”