Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) wa s the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymat ic activity, although it was 500-fold weaker than that of the pig heart ana log. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequ ence (SECIS). By performing appropriate nucleotide substitutions into the g ene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that af forded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxid ase. The recombinant enzyme, whose synthesis is selenium-dependent, display ed a 4-fold enhanced peroxidase activity as compared with the Cys-containin g analog, thus confirming the higher catalytic power of Sec compared with C ys in cit-PHGPx active site. The study led also to refinement of the minima l sequence requirements of the bacterial-type SECIS, and, for the first tim e, to the heterologous expression in Escherichia coli of a eukaryotic selen oprotein containing a SECIS in its open reading frame.