RESUMEN
Ergothioneine (EGT) is a sulfur-containing amino acid analog that is biosynthesized in fungi and bacteria, accumulated in plants, and ingested by humans where it is concentrated in tissues under oxidative stress. While the physiological function of EGT is not yet fully understood, EGT is a potent antioxidant in vitro. Here we report that oxidized forms of EGT, EGT-disulfide (ESSE) and 5-oxo-EGT, can be reduced by the selenoenzyme mammalian thioredoxin reductase (Sec-TrxR). ESSE and 5-oxo-EGT are formed upon reaction with biologically relevant reactive oxygen species. We found that glutathione reductase (GR) can reduce ESSE, but only with the aid of glutathione (GSH). The reduction of ESSE by TrxR was found to be selenium dependent, with non-selenium-containing TrxR enzymes having little or no ability to reduce ESSE. In comparing the reduction of ESSE by Sec-TrxR in the presence of thioredoxin to that of GR/GSH, we find that the glutathione system is 10-fold more efficient, but Sec-TrxR has the advantage of being able to reduce both ESSE and 5-oxo-EGT directly. This represents the first discovered direct enzymatic recycling system for oxidized forms of EGT. Based on our in vitro results, the thioredoxin system may be important for EGT redox biology and requires further in vivo investigation.
RESUMEN
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 ß-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.
