Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase.
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.
Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012, USA.
Matt D
MD
Wolfe
Farzana
F
Ahmed
Gerard M
GM
Lacourciere
Charles T
CT
Lauhon
Thressa C
TC
Stadtman
Timothy J
TJ
Larson
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000502:physiology*; D012643:Selenium / Q000378:metabolism; D018036:Selenium Compounds; D013466:Sulfurtransferases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000502:physiology
D029968:Escherichia coli Proteins; D010710:Phosphates; D018036:Selenium Compounds; C076818:selenophosphate; D013466:Sulfurtransferases; C088823:tRNA 2-selenouridine synthase; D013884:Thiosulfate Sulfurtransferase; D012643:Selenium