Selenoglutaredoxin as a Glutathione Peroxidase Mimic

Abstract

Glutaredoxin (Grx1) from Escherichia coli is a monomeric, 85-amino-acid-long, disulfide-containing redox protein. A Grx1 variant in which the redox-active disulfide was replaced with a selenocysteine (C11U/C14S) was prepared by native chemical ligation from three fragments as a potential mimic of the natural selenoenzyme glutathione peroxidase (Gpx). Selenoglutaredoxin, like the analogous C14S Grx1 variant, shows weak peroxidase activity. The selenol provides a 30-fold advantage over the thiol, but its activity is four orders of magnitude lower than that of bovine Gpx. In contrast, selenoglutaredoxin is an excellent catalyst for thiol-disulfide exchange reactions; it promotes the reduction of beta-hydroxyethyldisulfide by glutathione with a specific activity of 130 units mg(-1). This value is 1.8 times greater than that of C14S Grx1 under identical conditions, and >10(4) greater than the peroxidase activity of either enzyme. Given the facile reduction of the glutathionyl-selenoglutaredoxin adduct by glutathione, oxidation of the selenol by the alkyl hydroperoxide substrate likely limits catalytic turnover and will have to be optimized to create more effective Gpx mimics. These results highlight the challenge of generating Gpx activity in a small, generic protein scaffold, despite the presence of a well-defined glutathione binding site and the intrinsic advantage of selenium over sulfur derivatives.

Selenocysteine in Thiol/Disulfide-Like Exchange Reactions

“…This property has experimental support; the substitution of Sec in place of Ser in subtilisin converted the enzyme into a peroxidase (89). The substitution of Sec in place of Cys in glutaredoxin also conferred the mutant enzyme with peroxidase activity (12).…”

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

“…More generally, it represents a valuable spectroscopic and mechanistic probe (7) and has been used to create artificial catalysts with tailored hydrolytic and redox properties (9)(10)(11)(41)(42)(43)(44). A robust method for placing selenocysteine at any site, in any protein would greatly enhance its utility.…”

Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation

“…This difference makes direct selenoprotein expression from mammalian selenoprotein-encoding genes in E. coli impossible (26). Alternative approaches for production of selenoproteins include chemical methods (14,15,27) or the utili-zation of novel tRNA species compatible with elongation factor Tu that thereby bypass requirements for SelB and SECIS elements in E. coli (28 -32). Yields and specificities in Sec insertion using these methods, however, still require improvements.…”