Ancient Loss of Catalytic Selenocysteine Spurred Convergent Adaptation in a Mammalian Oxidoreductase.

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical-chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions.

An efficient selenium transport pathway of selenoprotein P utilizing a high-affinity ApoER2 receptor variant and being independent of selenocysteine lyase.

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.

Molecular Mechanisms of the Cytotoxic Effect of Recombinant Selenoprotein SELENOM on Human Glioblastoma Cells.

Currently, selenobiology is an actively developing area, primarily due to the study of the role of the trace element selenium and its organic and inorganic compounds in the regulation of vital processes occurring in the cell. In particular, the study of the functions of selenium nanoparticles has gained great popularity in recent years. However, a weak point in this area of biology is the study of the functions of selenoproteins, of which 25 have been identified in mammals to date. First of all, this is due to the difficulties in obtaining native forms of selenoproteins in preparative quantities, due to the fact that the amino acid selenocysteine is encoded by one of the three stop codons of the TGA universal genetic code. A complex system for recognizing a given codon as a selenocysteine codon has a number of features in pro- and eukaryotes. The selenoprotein SELENOM is one of the least studied mammalian selenoproteins. In this work, for the first time, studies of the molecular mechanisms of regulation of the cytotoxic effect of this protein on human glioblastoma cells were carried out. The cytotoxicity of cancer cells in our experiments was already observed when cells were exposed to 50 µg of SELENOM and increased in proportion to the increase in protein concentration. Apoptosis of human glioblastoma cells was accompanied by an increase in mRNA expression of a number of pro-apoptotic genes, an increase in endoplasmic reticulum stress, and activation of the UPR IRE1α signaling pathway. The results obtained also demonstrate a dose-dependent depletion of the Ca2+ pool under the action of SELENOM, which proves the important role of this protein in the regulation of calcium homeostasis in the cell.

Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4 µg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.

Selenium and breast cancer - An update of clinical and epidemiological data.

There is an urgent need for new and improved therapeutic strategies in breast cancer, which is the most common malignancy affecting women in the United States and worldwide. Selenium (Se) is an essential trace element of the human diet and plays a critical role in many aspects of human health. Clinical and epidemiological studies summarized here clearly demonstrate that Se status correlates with breast cancer survival. As a result, one way to curb breast cancer mortality would be via Se supplementation, especially in patients with severely deplete Se status. Se manifests its biological activity through incorporation into selenoproteins as selenocysteine. However, a better understanding of tissue-specific mechanisms and roles for selenoproteins in general is required. Additionally, many human selenoproteins harbor single nucleotide polymorphisms, which impact protein expression and activity and have been associated with cancer susceptibility or impacting survival. Increasing evidence indicates that these genetic variations impinge on the interactions between Se and breast cancer. This highlights the importance of integrating the Se status with genetic factors to fully define the benefit of Se in breast cancer. While Se supplementation would clearly benefit a subset of patients, this requires first the identification of at-risk patients and warrants validation through intervention trials.

The selenocysteine toolbox: A guide to studying the 21st amino acid.

Selenocysteine (Sec), the 21st genetically encoded amino acid, is structurally similar to cysteine (Cys) but with a sulfur to selenium replacement. This small change confers Sec with related chemical properties to Cys but often with enhanced reactivity. In organisms, Sec is present in selenoproteins taking on various roles such as cellular maintenance, immune response, hormone regulation, and oxidative stress. The detailed reactions of Sec in these functions remains unclear and has been a difficult question to answer. This is related to the low natural expression of selenoproteins and their complicated biosynthesis pathway. As a result, the focus in selenoprotein research has been on the expansion of tools and techniques to promote research in this area. Over the past two decades there has been immense progress in the development of selenoprotein expression systems, Sec-detection methods, and genomic databases. In this review we have compiled these tools systematically, highlighting their strengths and clarifying the limitations, as a resource for future selenoprotein research.

Selenoproteins and the senescence-associated epitranscriptome.

Selenium is a naturally found trace element, which provides multiple benefits including antioxidant, anticancer, and antiaging, as well as boosting immunity. One unique feature of selenium is its incorporation as selenocysteine, a rare 21st amino acid, into selenoproteins. Twenty-five human selenoproteins have been discovered, and a majority of these serve as crucial antioxidant enzymes for redox homeostasis. Unlike other amino acids, incorporation of selenocysteine requires a distinctive UGA stop codon recoding mechanism. Although many studies correlating selenium, selenoproteins, aging, and senescence have been performed, it has not yet been explored if the upstream events regulating selenoprotein synthesis play a role in senescence-associated pathologies. The epitranscriptomic writer alkylation repair homolog 8 (ALKBH8) is critical for selenoprotein production, and its deficiency can significantly decrease levels of selenoproteins that are essential for reactive oxygen species (ROS) detoxification, and increase oxidative stress, one of the major drivers of cellular senescence. Here, we review the potential role of epitranscriptomic marks that govern selenocysteine utilization in regulating the senescence program.

Selenocysteine Machinery Primarily Supports TXNRD1 and GPX4 Functions and Together They Are Functionally Linked with SCD and PRDX6.

The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD.

Expression of selenoproteins via genetic code expansion in mammalian cells.

Selenoproteins, which contain the 21st amino acid selenocysteine, play roles in maintaining cellular redox homeostasis. Many open questions remain in the field of selenoprotein biology, including the functions of a number of uncharacterized human selenoproteins, and the properties of selenocysteine compared to its analogous amino acid cysteine. The mechanism of selenocysteine incorporation involves an intricate machinery that deviates from the mechanism of incorporation for the canonical 20 amino acids. As a result, recombinant expression of selenoproteins has been historically challenging, and has hindered a deeper evaluation of selenoprotein biology. Genetic code expansion methods, which incorporate protected analogs of selenocysteine, allow the endogenous selenocysteine incorporation mechanism to be bypassed entirely to facilitate selenoprotein expression. Here we present a method for incorporating a photocaged selenocysteine amino acid (DMNB-Sec) into human selenoproteins directly in mammalian cells. This approach offers the opportunity to study human selenoproteins in their native cellular environment and should advance our understanding of selenoprotein biology.

Intein-based Design Expands Diversity of Selenocysteine Reporters.

The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts.

The Possible Mechanism of Physiological Adaptation to the Low-Se Diet and Its Health Risk in the Traditional Endemic Areas of Keshan Diseases.

Selenium is an essential trace element for humans and animals. As with oxygen and sulfur, etc., it belongs to the sixth main group of the periodic table of elements. Therefore, the corresponding amino acids, such as selenocysteine (Sec), serine (Ser), and cysteine (Cys), have similar spatial structure, physical, and chemical properties. In this review, we focus on the neglected but key role of serine in a possible mechanism of the physiological adaptation to Se-deficiency in human beings with an adequate intake of dietary protein: the insertion of Cys in place of Sec during the translation of selenoproteins dependent on the Sec insertion sequence element in the 3'UTR of mRNA at the UGA codon through a novel serine-dependent pathway for the de novo synthesis of the Cys-tRNA[Ser]Sec, similar to Sec-tRNA[Ser]Sec. We also discuss the important roles of serine in the metabolism of selenium directly or indirectly via GSH, and the maintenance of selenium homostasis regulated through the methylation modification of Sec-tRNA[Ser]Sec at the position 34U by SAM. Finally, we propose a hypothesis to explain why Keshan disease has gradually disappeared in China and predict the potential health risk of the human body in the physiological adaptation state of low selenium based on the results of animal experiments.

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2'O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance.

In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)2 fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent KMH2 values, do not produce H2 at pH 6, and display the same onset overpotential for H2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O2 tolerance (apparent KMH2 and Vmax [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H-, H2, O2) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H2 oxidation by this enzyme.

Introducing Selenocysteine into Recombinant Proteins in Escherichia coli.

Selenoproteins contain the 21st amino acid, selenocysteine. Selenocysteine is the only amino acid that is synthesized on its cognate tRNA, and it is inserted at specific recoded UGA stop codons via a complex translation system. Although highly similar to cysteine, selenocysteine has unique properties, including a stronger nucleophilic ability and lower reduction potential. Efforts to site-specifically incorporate selenocysteine to create recombinant selenoproteins involve a recoded UAG stop codon and expression of the necessary selenocysteine translation machinery. This article presents a protocol for expressing and purifying selenoproteins in Escherichia coli. © 2021 Wiley Periodicals LLC. Basic Protocol: Recombinant selenoprotein production in E. coli using a rewired translation system.

Quantitative Proteomics Reveals UGA-Independent Misincorporation of Selenocysteine throughout the Escherichia coli Proteome.

Selenocysteine is cotranslationally inserted into polypeptide chains by recoding the stop codon UGA. However, selenocysteine has also been found to be misincorporated into a small number of proteins displacing cysteines in previous studies, but such misincorporation has not yet been examined at the proteome level thoroughly. We performed label-free quantitative proteomics analysis on Escherichia coli grown in a high-selenium medium to obtain a fuller picture of selenocysteine misincorporation in its proteome. We found 139 misincorporation sites, including 54 recurred in all biological replicates, suggesting that some cysteine sites are more prone to be misincorporated than others. However, sequence and evolutionary conservation analysis showed no clear pattern among these misincorporation sites. We hypothesize that misincorporations occur randomly throughout the proteome, but the degradation rate of such misincorporated proteins varies depending on the impact of the misincorporation on protein function and stability, leading to the differential detectability of misincorporated sites by proteomics. Our hypothesis is further supported by two observations: (1) cells cultured with severely limited sulfur still retained a substantial proportion of normal cysteine counterparts of all of the found misincorporated proteins and (2) proteins involved in protein folding and proteolysis were highly upregulated in high-selenium culture.

Can Selenoenzymes Resist Electrophilic Modification? Evidence from Thioredoxin Reductase and a Mutant Containing α-Methylselenocysteine.

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.

SBP2 deficiency in adipose tissue macrophages drives insulin resistance in obesity.

Proinflammatory activation and accumulation of adipose tissue macrophages (ATMs) are associated with increased risk of insulin resistance in obesity. Here, we described the previously unidentified role of selenocysteine insertion sequence-binding protein 2 (SBP2) in maintaining insulin sensitivity in obesity. SBP2 was suppressed in ATMs of diet-induced obese mice and was correlated with adipose tissue inflammation. Loss of SBP2 initiated metabolic activation of ATMs, inducing intracellular reactive oxygen species content and inflammasome, which subsequently promoted IL-1ß-associated local proliferation and infiltration of proinflammatory macrophages. ATM-specific knockdown of SBP2 in obese mice promoted insulin resistance by increasing fat tissue inflammation and expansion. Reexpression of SBP2 improved insulin sensitivity. Last, an herbal formula that specifically induced SBP2 expression in ATMs can experimentally improve insulin sensitivity. Clinical observation revealed that it improved hyperglycemia in patients with diabetes. This study identified SBP2 in ATMs as a potential target in rescuing insulin resistance in obesity.

S-Glutathionylation of mouse selenoprotein W prevents oxidative stress-induced cell death by blocking the formation of an intramolecular disulfide bond.

Mouse selenoprotein W (SELENOW) is a small protein containing a selenocysteine (Sec, U) and four cysteine (Cys, C) residues. The Sec residue in SELENOW is located within the conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin (Trx). It is known that glutathione (GSH) binds to SELENOW and that this binding is involved in protecting cells from oxidative stress. However, the regulatory mechanisms controlling the glutathionylation of SELENOW in oxidative stress are unclear. In this study, using purified recombinant SELENOW in which Sec13 was changed to Cys, we found that SELENOW was glutathionylated at Cys33 and that this S-glutathionylation was enhanced by oxidative stress. We also found that the S-glutathionylation of SELENOW at Cys33 in HEK293 cells was due to glutathione S-transferase Pi (GSTpi) and that this modification was reversed by glutaredoxin1 (Grx1). In addition to the disulfide bond between the Cys10 and Cys13 of SELENOW, a second disulfide bond was formed between Cys33 and Cys87 under oxidative stress conditions. The second disulfide bond was reduced by Trx1, but the disulfide bond between Cys10 and Cys13 was not. The second disulfide bond was also reduced by glutathione, but the disulfide bond in the CXXC motif was not. The second disulfide bond of the mutant SELENOW, in which Cys37 was replaced with Ser, was formed at a much lower concentration of hydrogen peroxide than the wild type. We also observed that Cys37 was required for S-glutathionylation, and that S-glutathionylated SELENOW containing Cys37 protected the cells from oxidative stress. Furthermore, the SELENOW (C33, 87S) mutant, which could not form the second disulfide bond, also showed antioxidant activity. Taken together, these results indicate that GSTpi-mediated S-glutathionylation of mouse SELENOW at Cys33 is required for the protection of cells in conditions of oxidative stress, through inhibition of the formation of the second disulfide bond.

Gain of function conferred by selenocysteine: catalytic enhancement of one-electron transfer reactions by thioredoxin reductase.

Selenocysteine (Sec) is the 21st amino acid in the genetic code and it is present in a small number of proteins where it replaces the much more commonly used amino acid cysteine (Cys). It is both more complicated and bioenergetically costly to insert Sec into a protein in comparison to Cys, and this cost is most likely compensated by a gain of function to the enzyme/protein in which it is incorporated. Here we investigate one such gain of function, the enhancement of one-electron transfer catalysis. We compared the ability of Sec-containing mouse mitochondrial thioredoxin reductase (mTrxR2) to catalyze the reduction of bovine cytochrome c, ascorbyl radical, and dehydroascorbate in comparison to Cys-containing thioredoxin reductases from D. melanogaster (DmTrxR) and P. falciparum (PfTrxR). The Sec-containing mTrxR2 was able to reduce all three substrates, while the Cys-containing enzymes had little or no activity. In addition, we constructed Cys➔Sec mutants of DmTrxR and PfTrxR and found that this substitution resulted in a gain of function, as these mutant enzymes were now able to catalyze the reduction of these substrates. We also found that in the case of PfTrxR, reduction of cytochrome c was enhanced five-fold in a truncated PfTrxR in which the C-terminal redox center was removed. This shows that some of the ability of thioredoxin reductase to reduce this substrate comes from the flavin coenzyme. We also discuss a possible mechanism by which Sec-containing thioredoxin reductase reduces dehydroascorbate to ascorbate by two sequential, one-electron reductions, in part catalyzed by Sec.

The unique tRNASec and its role in selenocysteine biosynthesis.

Selenium (Se) is an essential trace element for several organisms and is mostly present in proteins as L-selenocysteine (Sec or U). Sec is synthesized on its L-seryl-tRNASec to produce Sec-tRNASec molecules by a dedicated selenocysteine synthesis machinery and incorporated into selenoproteins at specified in-frame UGA codons. UGA-Sec insertion is signaled by an mRNA stem-loop structure called the SElenoCysteine Insertion Sequence (SECIS). tRNASec transcription regulation and folding have been described showing its importance to Sec biosynthesis. Here, we discuss structural aspects of Sec-tRNASec and its role in Sec biosynthesis as well as Sec incorporation into selenoproteins. Defects in the Sec biosynthesis or incorporation pathway have been correlated with pathological conditions.