NAD-dependent RNA terminal 2' and 3' phosphomonoesterase activity of a subset of Tpt1 enzymes.

The enzyme Tpt1 removes the 2'-PO 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 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 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. and Tpt1 proteins are active on 2'-PO and 3'-PO ends, with a 2.4- to 2.6-fold kinetic preference for the 2'-PO 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. Tpt1 acts specifically on a terminal 2'-PO end and not with a 3'-PO In contrast, Tpt1 and human Tpt1 are ineffective in removing either a 2'-PO or 3'-PO end.