RESUMO
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 tRNALys (S2U-RNA) and recombinant SelU with an MBP tag, we found that only geranyl (C10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C5), dimethylallyl (C5), farnesyl (C15) and geranylgeranyl (C20) are not. Interestingly, methyl (C1)- and C5-, C10-, and C15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by SelU, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl ≥ farnesyl > dimethylallyl â« 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 (ΔG) 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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Selênio , Sulfurtransferases , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Neopreno , Sulfurtransferases/genética , Sulfurtransferases/metabolismoRESUMO
To date the only tRNAs containing nucleosides modified with a selenium (5-carboxymethylaminomethyl-2-selenouridine and 5-methylaminomethyl-2-selenouridine) have been found in bacteria. By using tRNA anticodon-stem-loop fragments containing S2U, Se2U, or geS2U, we found that in vitro tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA in a two-step process involving S2U-RNA geranylation (with ppGe) and subsequent selenation of the resulting geS2U-RNA (with SePO33- ). No 'direct' S2U-RNAâSe2U-RNA replacement is observed in the presence of SelU/SePO33- only (without ppGe). These results suggest that the in vivo S2UâSe2U and S2UâgeS2U transformations in tRNA, so far claimed to be the elementary reactions occurring independently in the same domain of the SelU enzyme, should be considered a combination of two consecutive events - geranylation (S2UâgeS2U) and selenation (geS2UâSe2U).
Assuntos
Escherichia coli/enzimologia , Compostos Organosselênicos/metabolismo , Selênio/metabolismo , Sulfurtransferases/fisiologia , Terpenos/metabolismo , Uridina/análogos & derivados , Sítios de Ligação , Carbono/metabolismo , Catálise , Escherichia coli/genética , Fosfatos de Poli-Isoprenil/metabolismo , Processamento de Proteína Pós-Traducional/genética , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Sulfurtransferases/genética , Tiouridina/química , Tiouridina/metabolismo , Uridina/metabolismoRESUMO
Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.