Functional Diversity of the Rhodanese Homology Domain THE ESCHERICHIA COLI ybbB GENE ENCODES A SELENOPHOSPHATE-DEPENDENT tRNA 2-SELENOURIDINE SYNTHASE

Abstract 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 (mnm5se2U), is located at the wobble position of the anticodons of tRNALys, tRNAGlu, and tRNA1Gln. Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm5se2U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm5se2U 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 His6 tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm5se2U synthesis is shown to be dependent on 2-selenouridine synthase, SePO3, and tRNA. Finally, we demonstrate that the conserved Cys97 (but not Cys96) in the rhodanese sequence motif Cys96-Cys97-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.