Structure–function analysis of an ancient TsaD–TsaC–SUA5–TcdA modular enzyme reveals a prototype of tRNA t(6)A and ct(6)A synthetases

N (6)-threonylcarbamoyladenosine (t(6)A) is a post-transcriptional modification found uniquely at position 37 of tRNAs that decipher ANN-codons in the three domains of life. tRNA t(6)A plays a pivotal role in promoting translational fidelity and maintaining protein homeostasis. The biosynthesis of tRNA t(6)A requires members from two evolutionarily conserved protein families TsaC/Sua5 and TsaD/Kae1/Qri7, and a varying number of auxiliary proteins. Furthermore, tRNA t(6)A is modified into a cyclic hydantoin form of t(6)A (ct(6)A) by TcdA in bacteria. In this work, we have identified a TsaD–TsaC–SUA5–TcdA modular protein (TsaN) from Pandoraviruses and determined a 3.2 Å resolution cryo-EM structure of P. salinus TsaN. The four domains of TsaN share strong structural similarities with TsaD/Kae1/Qri7 proteins, TsaC/Sua5 proteins, and Escherichia coli TcdA. TsaN catalyzes the formation of threonylcarbamoyladenylate (TC-AMP) using L-threonine, HCO(3)(−) and ATP, but does not participate further in tRNA t(6)A biosynthesis. We report for the first time that TsaN catalyzes a tRNA-independent threonylcarbamoyl modification of adenosine phosphates, leading to t(6)ADP and t(6)ATP. Moreover, TsaN is also active in catalyzing tRNA-independent conversion of t(6)A nucleoside to ct(6)A. Our results imply that TsaN from Pandoraviruses might be a prototype of the tRNA t(6)A- and ct(6)A-modifying enzymes in some cellular organisms.