NAD(+)-dependent RNA terminal 2′ and 3′ phosphomonoesterase activity of a subset of Tpt1 enzymes

The enzyme Tpt1 removes the 2′-PO(4) at the splice junction generated by fungal tRNA ligase; it does so via a two-step reaction in which (i) the internal RNA 2′-PO(4) attacks NAD(+) to form an RNA-2′-phospho-ADP-ribosyl intermediate; and (ii) transesterification of the ribose O2″ to the 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. The role that Tpt1 enzymes play in taxa that have no fungal-type RNA ligase remains obscure. An attractive prospect is that Tpt1 enzymes might catalyze reactions other than internal RNA 2′-PO(4) removal, via their unique NAD(+)-dependent transferase mechanism. This study extends the repertoire of the Tpt1 enzyme family to include the NAD(+)-dependent conversion of RNA terminal 2′ and 3′ monophosphate ends to 2′-OH and 3′-OH ends, respectively. The salient finding is that different Tpt1 enzymes vary in their capacity and positional specificity for terminal phosphate removal. Clostridium thermocellum and Aeropyrum pernix Tpt1 proteins are active on 2′-PO(4) and 3′-PO(4) ends, with a 2.4- to 2.6-fold kinetic preference for the 2′-PO(4). The accumulation of a terminal 3′-phospho-ADP-ribosylated RNA intermediate during the 3′-phosphotransferase reaction suggests that the geometry of the 3′-p-ADPR adduct is not optimal for the ensuing transesterification step. Chaetomium thermophilum Tpt1 acts specifically on a terminal 2′-PO(4) end and not with a 3′-PO(4). In contrast, Runella slithyformis Tpt1 and human Tpt1 are ineffective in removing either a 2′-PO(4) or 3′-PO(4) end.