Abstact

Tpt1/TRPT1/KptA family proteins are evolutionarily conserved in all three domains of life. In fungi and plants, Tpt1 transfers 2'-PO(4)(2-) from tRNA splice junction to NAD(+), which is the final step of tRNA maturation and is critical for the function of tRNA. In mammals and bacteria, Tpt1-catalyzed reaction leads to 5'-end ADP ribosylation, a reversible chemical modification of nucleic acids. Based on in vivo and in vitro biochemical studies, a two-step catalytic mechanism has been established for Tpt1-catalyzed RNA 2'-PO(4)(2-) transfer, including (i) the 2'-PO(4)(2-) attacks NAD(+), releasing nicotinamide and forming a 2'-phospho-ADP-ribosylated RNA (2'-p-ADPR-RNA) intermediate; and (ii) transesterification of the ADP-ribose 2"-OH to RNA 2'-PO(4)(2-), displacing the 2'-OH RNA and producing ADP-ribose-1",2"-cyclic phosphate (Appr>P). However, neither 2'-p-ADPR-RNA intermediate nor Appr>P product has been captured in any reported Tpt1 structures. Here, we report a series of crystal structures of T. kodakarensis Tpt1 (TkoTpt1), capturing the key 2'-p-ADPR-RNA intermediate. In addition, our structures also capture the 5'-p-ADPR-DNA intermediate and Appr>P product. Structural analysis and in vitro catalytic assays revealed that TkoTpt1 utilizes similar mechanism in 2'-PO(4)(2-) and 5'-PO(4)(2-) transfer. In conclusion, our structures reaffirm the catalytic mechanism of Tpt1-catalyzed phosphate transfer.