Publications

• Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase.

  Wolfe M.D., Ahmed F., Lacourciere G.M., Lauhon C.T., Stadtman T.C., Larson T.J.

  Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism ... >> More

  Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes. << Less

  J. Biol. Chem. 279:1801-1809(2004) [PubMed] [EuropePMC]

• Transformation of a wobble 2-thiouridine to 2-selenouridine via S-geranyl-2-thiouridine as a possible cellular pathway.

  Bartos P., Maciaszek A., Rosinska A., Sochacka E., Nawrot B.

  The newly discovered S-geranylated 2-thiouridines (geS2U) (Dumelin et al., 2012) and 2-selenouridines (Se2U) were recently shown to be synthesized by a single enzyme (selenouridine synthase, SelU) through two distinct pathways using the same 2-thiouridine substrate (S2U); however, no clear catalyt ... >> More

  The newly discovered S-geranylated 2-thiouridines (geS2U) (Dumelin et al., 2012) and 2-selenouridines (Se2U) were recently shown to be synthesized by a single enzyme (selenouridine synthase, SelU) through two distinct pathways using the same 2-thiouridine substrate (S2U); however, no clear catalytic mechanism was proposed. We suggest that S-geranyl-2-thiouridine is an intermediate of the SelU-catalyzed conversion of S2U to Se2U. The successful chemical transformation of S2U→geS2U→Se2U is demonstrated here as an initial approximation of the intracellular pathway. The structure of Se2U was confirmed by spectroscopic methods, which included, for the first time, (77)Se NMR data (δ 354ppm). << Less

  Bioorg. Chem. 56:49-53(2014) [PubMed] [EuropePMC]

• Transfer RNA bound to MnmH protein is enriched with geranylated tRNA--a possible intermediate in its selenation?

  Jaeger G., Chen P., Bjoerk G.R.

  The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)-a reaction catalyzed by the seleno ... >> More

  The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)-a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s2 group of mnm5s2U and of cmnm5s2U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm5ges2U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH+ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm5s2U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA. << Less

  PLoS ONE 11:E0153488-E0153488(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Escherichia coli tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA via S-geranylated-intermediate.

  Sierant M., Leszczynska G., Sadowska K., Komar P., Radzikowska-Cieciura E., Sochacka E., Nawrot B.

  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-selenou ... >> More

  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 SePO₃^3- ). No 'direct' S2U-RNA→Se2U-RNA replacement is observed in the presence of SelU/SePO₃^3- only (without ppGe). These results suggest that the in vivo S2U→Se2U and S2U→geS2U 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 (S2U→geS2U) and selenation (geS2U→Se2U). << Less

  FEBS Lett. 592:2248-2258(2018) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• A purified selenophosphate-dependent enzyme from Salmonella typhimurium catalyzes the replacement of sulfur in 2-thiouridine residues in tRNAs with selenium.

  Veres Z., Stadtman T.C.

  A tRNA-modifying enzyme tentatively termed tRNA 2-selenouridine synthase was purified by a five-step procedure that resulted in 50-60% pure preparations. This enzyme catalyzes the conversion of a 5-methylaminomethyl-2-thiouridine residue in the tRNA substrate to 5-methylaminomethyl-2-selenouridine ... >> More

  A tRNA-modifying enzyme tentatively termed tRNA 2-selenouridine synthase was purified by a five-step procedure that resulted in 50-60% pure preparations. This enzyme catalyzes the conversion of a 5-methylaminomethyl-2-thiouridine residue in the tRNA substrate to 5-methylaminomethyl-2-selenouridine. The selenium donor substrate for this reaction is shown to be selenophosphate which is formed from ATP and selenide by selenophosphate synthetase. Replacement of sulfur with selenium in tRNAs catalyzed by tRNA 2-selenouridine synthase occurs in the absence of ATP. The dependence of reaction velocity on selenophosphate concentration obeys Michaelis-Menten kinetics indicating an apparent Km value of 17.1 microM. Bulk thio-tRNA preparations from Escherichia coli and Salmonella typhimurium are equally effective as substrates for the selenium incorporation reaction. An intact 3' end of the tRNA molecule does not seem to be essential for selenium incorporation. Identity of the product of the reaction was confirmed by HPLC analysis of digests of [75Se]seleno-tRNAs labeled by incubation with the purified enzyme. A labeled compound in the nucleoside mixture was coeluted with authentic 5-methylaminomethyl-2-selenouridine. << Less

  Proc. Natl. Acad. Sci. U.S.A. 91:8092-8096(1994) [PubMed] [EuropePMC]