Abstract
Selenocysteine (Sec) is the 21st proteogenic amino acid in the genetic code. Incorporation of Sec into proteins is a complex and bioenergetically costly process that evokes the following question: "Why did nature choose selenium?" An answer that has emerged over the past decade is that Sec confers resistance to irreversible oxidative inactivation by reactive oxygen species. Here, we explore the question of whether this concept can be broadened to include resistance to reactive electrophilic species (RES) because oxygen and related compounds are merely a subset of RES. To test this hypothesis, we inactivated mammalian thioredoxin reductase (Sec-TrxR), a mutant containing α-methylselenocysteine [(αMe)Sec-TrxR], and a cysteine ortholog TrxR (Cys-TrxR) with various electrophiles, including acrolein, 4-hydroxynonenal, and curcumin. Our results show that the acrolein-inactivated Sec-TrxR and the (αMe)Sec-TrxR mutant could regain 25% and 30% activity, respectively, when incubated with 2 mM H2O2 and 5 mM imidazole. In contrast, Cys-TrxR did not regain activity under the same conditions. We posit that Sec enzymes can undergo a repair process via β-syn selenoxide elimination that ejects the electrophile, leaving the enzyme in the oxidized selenosulfide state. (αMe)Sec-TrxR was created by incorporating the non-natural amino acid (αMe)Sec into TrxR by semisynthesis and allowed for rigorous testing of our hypothesis. This Sec derivative enables higher resistance to both oxidative and electrophilic inactivation because it lacks a backbone Cα-H, which prevents loss of selenium through the formation of dehydroalanine. This is the first time this unique amino acid has been incorporated into an enzyme and is an example of state-of-the-art protein engineering.