ArcS from Thermococcus kodakarensis transfers L-lysine to preQ₀ nucleoside derivatives as minimum substrate RNAs

Archaeosine (G⁺) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ₀). In Euryarchaea, preQ₀15 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ₀-lysine (preQ₀-Lys) as an intermediate. The resulting preQ₀-Lys15 in tRNA is converted to G⁺15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ₀ were determined using a purified enzyme. RNA fragments containing preQ₀ were prepared from Saccharomyces cerevisiae tRNA^Phe-preQ₀15. ArcS transferred ¹⁴C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ₀ nucleoside and preQ₀ nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ₀-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ₀-Lys15 in tRNA with guanine or preQ₀ base, showing that formation of tRNA-preQ₀-Lys by ArcS plays a role in preventing the reverse reaction in G⁺ biosynthesis.