Escherichia coli tRNA 2-selenouridine synthase SelU selects its prenyl substrate to accomplish its enzymatic function

Bacterial tRNA 2-selenouridine synthase (SelU) in vitro converts S2U-RNA to its selenium analog (Se2U-RNA) in a two-step process: (i) geranylation of S2U-RNA (with geranyl pyrophosphate, gePP), and (ii) selenation of the resulting geS2U-RNA (with the selenophosphate anion, SePO33-). Using an S2U-containing anticodon stem-loop fragment derived from tRNA(LYs) (S2U:I-RNA) and recombinant Selp with an MBP tag, we found that only geranyl (C-10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C-5), dimethylallyl (C-5), farnesyl (C-15) and geranylgeranyl (C-20) are not. Interestingly, methyl (C-1)- and C-5-, C-10-, and C-15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by Se1U, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl >= farnesyl > dimethylallyl >> methyl. Microscale thermophoresis showed an affinity between gePP and SelU in the micromolar range, while the other pyrophosphates tested, such as isopentenyl, dimethylallyl, farnesyl and geranylgeranyl, either did not bind to the protein or their binding affinity was above 1 mM. These results agree well with the in silico analysis, with gePP being the best binding substrate (the lowest relative free energy of binding (Delta G) and a small solvent-accessible surface area (SASA)). These results suggest that SelU has high substrate specificity for the prenylation reaction (only gePP is accepted), whereas there is little discrimination for the selenation reaction. We therefore suggest that only gePP and the geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs, as it is found in the bacterial system.

Automated summary

The bacterial tRNA 2-selenouridine synthase SelU shows high substrate specificity for geranyl pyrophosphate in the prenylation reaction, while there is little discrimination in the selenation reaction. It is suggested that only geranyl pyrophosphate and geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs in the bacterial system.