A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum

A strongly Se-75-labeled 22 kDa protein detected previously showed in its N -terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22 470 Da) and co ntains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cy steine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologou s expression of the Eubacterium acidaminophilum PrxU as a recombinant selen oprotein in Escherichia coli was not possible. A cysteine-encoding mutant g ene, prxU47C, containing UGC instead of UGA was strongly expressed. This re combinant PrxU47C mutant protein was purified to homogeneity by its affinit y tag, but was not active as a thiol-dependent peroxidase. The identificati on of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously onl y known as non-selenoproteins containing catalytic cysteine residues.