Structure–function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t⁶A tRNA modification

<jats:p><jats:italic>N</jats:italic><jats:sup>6</jats:sup>-threonyl-carbamoyl adenosine (t<jats:sup>6</jats:sup>A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t<jats:sup>6</jats:sup>A stabilizes the codon–anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t<jats:sup>6</jats:sup>A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure–function analysis of <jats:italic>Pyrococcus abyssi</jats:italic> Sua5 (<jats:italic>Pa</jats:italic>-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of <jats:italic>Pa</jats:italic>-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain–domain interface are essential for the function of <jats:italic>Pa</jats:italic>-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the <jats:italic>N</jats:italic>-carboxy-<jats:sc>l</jats:sc>-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.</jats:p>