No Selenium Required: Reactions Catalyzed by Mammalian Thioredoxin Reductase That Are Independent of a Selenocysteine Residue

Abstract

Mammalian thioredoxin reductase (TR) contains a rare selenocysteine (Sec) residue in a conserved redox active tetrapeptide of sequence Gly-Cys1-Sec2-Gly. The high chemical reactivity of the Sec residue is thought to confer broad substrate specificity to the enzyme. In addition to utilizing thioredoxin (Trx) as a substrate, other substrates are: protein disulfide isomerase, glutaredoxin, glutathione peroxidase, NK-lysin/granulsin, HIV Tat protein, H2O2, lipid hydroperoxides, vitamin K, ubiquinone, juglone, ninhydrin, alloxan, dehydroascorbate, DTNB, lipoic acid/lipoamide, S-nitrosoglutathione, selenodiglutathione, selenite, methylseleninate, and selenocystine. Here we show that the Cys2-mutant enzyme or the N- terminal reaction center alone can reduce Se-containing substrates selenocystine and selenite with only slightly less activity than the wild type enzyme, in stark contrast to when Trx is used as the substrate when the enzyme suffers a 175- to 550-fold reduction in kcat. Our data supports the use of alternative mechanistic pathways for the Se-containing substrates that bypass a critical ring-forming step when Trx is the substrate. We also show that lipoic acid can be reduced through a Sec-independent mechanism that involves the N-terminal reaction center. These results show that the broad substrate specificity of the mammalian enzyme is not due to the presence of the rare Sec residue, but is due to the catalytic power of the N-terminal reaction center. We hypothesize that the N-terminal reaction center can reduce substrates: (i) with good leaving groups such as DTNB, (ii) that are highly electrophilic such as selenite, (iii) or are activated by strain such as lipoic acid/lipoamide. We also show that the absence of Sec only changed the IC50 for aurothioglucose by a factor of 1.7 in the full-length mammalian enzyme (83 nM to 142 nM), but surprisingly the truncated enzyme showed much stronger inhibition (25 nM). This contrasts with auranofin, where the absence of Sec more strongly perturbed inhibition.

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

“…However, some substrates (e.g. selenocystine, selenite, and lipoic acid) are perhaps not solely dependent on the Sec and may alternatively be reduced by the N-terminal dithiol, at least for the mitochondrial isoenzyme TrxR2 (10). When the enzyme is reduced by NADPH, the nucleophilic and * This work was supported, in whole or in part, by National Institutes of Health Grant R01ES012707 (to C. R. M.).…”

“…Interestingly, different gold compounds may bind to different sites. For example, inhibition by auranofin is strongly associated with the Sec, whereas aurothioglucose is less Sec-dependent and may have some effects on the N-terminal dithiol (10). The N-terminal domains, including the flavin and Cys 59 /Cys 64 dithiol of TrxR, resemble those in glutathione reductase and lipoamide dehydrogenase, but only TrxR has the C-terminal Cys-Sec motif.…”

Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration

“…Thus, inhibition of TrxRs, in particular of the mitochondrial isoform, can alter the mitochondrial membrane permeability and induce the release of segregated proapoptotic factors, ultimately triggering cancer cell apoptosis (Gandin et al, 2010;Marzano et al, 2007). Yet, contrasting reports have appeared on this issue and no conclusive consensus has been reached on the "true" mode of action of AF (Rigobello et al, 2008;Omata et al, 2006;Lothrop et al, 2009). In fact, increased ROS production is not a common feature for AF treated cells; indeed, Rigobello et al (Rigobello et al, 2008) showed that, in Jurkat T cells, AF causes apoptosis with very limited oxidative stress and Omata et al (Omata et al, 2006) proposed a ROS-independent inhibition of the mitochondrial activity as principally responsible for the pro-apoptotic effects of AF.…”

“…In fact, increased ROS production is not a common feature for AF treated cells; indeed, Rigobello et al (Rigobello et al, 2008) showed that, in Jurkat T cells, AF causes apoptosis with very limited oxidative stress and Omata et al (Omata et al, 2006) proposed a ROS-independent inhibition of the mitochondrial activity as principally responsible for the pro-apoptotic effects of AF. Furthermore, the presence of a rare Sec residue in mammalian Thioredoxins, frequently cited as a main reason for the broad substrate enzyme specificity and as a target of gold(I) compounds, was recently discussed by Lothrop et al (Lothrop et al, 2009). Interestingly, these authors found that the truncated variant, TrxR2 missing the Sec residue, still reduced DTNB almost as efficiently as full-length TrxR2 and that a few gold(I) compounds, such as AF and aurothioglucose, effectively inhibited both the full-length enzymes and the TrxR2 enzyme.…”

DNA Replication in Animal Systems Lacking Thioredoxin Reductase I

“…However, the testis-specific mammalian TrxR3 protein, also called thioredoxinglutathione reductase or TGR, as well as the Sec-lacking TrxR protein in Drosophila, does reduce GSSG (Gromer et al, 2003;Johansson et al, 2006;Sun et al, 2001;Sun et al, 2005), and in some lower metazoans such as Echinococcus, a single gene, within the metazoan TrxR family and containing Sec in its C-terminal active site, encodes all known Trx-and GSSGreductase activities (Bonilla et al, 2008). One might hypothesize that, by exchanging the ancestral E. coli-type TrxR enzymes for the metazoan version, the evolutionary capacity of the lineage might have been potentiated; however since both enzymes will effectively reduce Trx, this model suggests the evolutionary advantage of the new enzyme for metazoans is related to other activities that differ between these enzyme types (Arner and Holmgren, 2000;Arner et al, 1996;Lothrop et al, 2009). Alternatively, one might imagine that, if an ancestral metazoan evolved a TGR enzyme that could replace both the ancestral TrxR and Gsr activities, as the Echinococcus version does, then perhaps the ancestral E. colitype TrxR and the ancestral Gsr were simply and irrevocably lost as being redundant with the new bi-functional TGR enzyme, as seen in Echinococcus.…”