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Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2′-PO 4  from the splice junction to NAD +  to form ADP-ribose 1″-2″cyclic phosphate and nicotinamide. To understand the structural requirements for  Saccharomyces cerevisiae  Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues—Arg23, His24, Arg71, and Arg138—as essential for Tpt1 function in vivo. Structure–activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the  Escherichia coli  ortholog KptA in complementing  tpt1 Δ was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30°C. These findings attest to the structural and functional conservation of Tpt1-like 2′-phosphotransferases and identify likely constituents of the active site.

Structure–function analysis of the yeast NAD+-dependent tRNA 2′-phosphotransferase Tpt1

Structure–function analysis of the yeast NAD⁺-dependent tRNA 2′-phosphotransferase Tpt1