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 (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.

Highlights

  • From the ‡Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8012, the ¶Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, and the **School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706

  • Certain ThiI proteins, including that of E. coli, contain a C-terminal rhodanese homology domain fused to an N-terminal domain that is involved in biosynthesis of the thiazole ring of thiamin and 4-thiouridine of tRNAs [15,16,17,18]

  • These results indicate that the rhodanese homology domain functions in selenium transfer from selenophosphate during conversion of 2-thiouridine to 2-selenouridine in bacterial tRNA

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Summary

Functional Diversity of the Rhodanese Homology Domain

THE ESCHERICHIA COLI ybbB GENE ENCODES A SELENOPHOSPHATE-DEPENDENT tRNA 2SELENOURIDINE SYNTHASE*□S. We demonstrate that the conserved Cys (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. The second cysteine residue (Cys97) of 2-selenouridine synthase that aligns most closely with the conserved active site cysteine of the rhodanese homology domain was found to be essential for activity in vivo. These results indicate that the rhodanese homology domain functions in selenium transfer from selenophosphate during conversion of 2-thiouridine to 2-selenouridine in bacterial tRNA

EXPERIMENTAL PROCEDURES
Derivation or reference
RESULTS
Without ATP
Wild type
DISCUSSION
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