Abstract
Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic (75)Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.
Highlights
Selenium is an important biological trace element
The two Sec in SelL were suggested to participate in redox catalysis through a diselenide bond (24), and even several Sec residues in SelP correspond to Cys in other homologs, including one Sec that corresponds to Cys in the catalytic sites of thiol oxidoreductases
It is widely accepted that new Sec residues primarily evolve to substitute Cys residues to carry out redox catalysis
Summary
Searches for EST Sequences with Dual SECIS Elements—The EST database “est_others” was downloaded from the NCBI; it contained 51,484,119 sequences (29,098,406,053 bp). Expression, and Purification—M. senile MsrB forms including either TGC for Cys or TCC for Ser in place of the TGA codon were transferred to the pET28a vector (Novagen, Madison, WI) without SECIS elements, and these recombinant vectors were transformed into Escherichia coli BL21 (DE3) cells for protein expression. Recombinant MsrB1Cys in the pET21b vector was transformed into E. coli BL21 cells (29) These cells were grown in LB medium containing 100 g/ml ampicillin until A600 reached 0.5, and isopropyl-1thio--D-galactopyranoside was added to a final concentration of 0.5 mM. Cell pellets were mixed with the resuspension buffer (50 mM Tris-HCl, pH 7.5, 15 mM imidazole, 300 mM NaCl), and protease inhibitor mixture (Roche Applied Science, Basel, Switzerland) was added to this mixture.
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