A hydantoin isoform of cyclic N(6)-threonylcarbamoyladenosine (ct(6)A) is present in tRNAs

N(6)-Threonylcarbamoyladenosine (t(6)A) and its derivatives are universally conserved modified nucleosides found at position 37, 3΄ adjacent to the anticodon in tRNAs responsible for ANN codons. These modifications have pleiotropic functions of tRNAs in decoding and protein synthesis. In certain species of bacteria, fungi, plants and protists, t(6)A is further modified to the cyclic t(6)A (ct(6)A) via dehydration catalyzed by TcdA. This additional modification is involved in efficient decoding of tRNA(Lys). Previous work indicated that the chemical structure of ct(6)A is a cyclic active ester with an oxazolone ring. In this study, we solved the crystal structure of chemically synthesized ct(6)A nucleoside. Unexpectedly, we found that the ct(6)A adopted a hydantoin isoform rather than an oxazolone isoform, and further showed that the hydantoin isoform of ct(6)A was actually present in Escherichia coli tRNAs. In addition, we observed that hydantoin ct(6)A is susceptible to epimerization under mild alkaline conditions, warning us to avoid conventional deacylation of tRNAs. A hallmark structural feature of this isoform is the twisted arrangement of the hydantoin and adenine rings. Functional roles of ct(6)A37 in tRNAs should be reconsidered.