Why do proteins use selenocysteine instead of cysteine?

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.     

Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.     

Differing views of the role of selenium in thioredoxin reductase

This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pK _a and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO₂ ⁻, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO₂ ⁻ to Cys-SH).     

Analysis of the E. coli NifS CsdB protein at 2.0 A reveals the structural basis for perselenide and persulfide intermediate formation

The Escherichia coli NifS CsdB protein is a member of the homodimeric pyridoxal 5'-phosphate (PLP)-dependent family of enzymes. These enzymes are capable of decomposing cysteine or selenocysteine into L-alanine and sulfur or selenium, respectively. E. coli NifS CsdB has a high specificity for L-selenocysteine in comparison to l-cysteine, suggesting a role for this enzyme is selenium metabolism. The 2.0 A crystal structure of E. coli NifS CsdB reveals a high-resolution view of the active site of this enzyme in apo-, persulfide, perselenide, and selenocysteine-bound intermediates, suggesting a mechanism for the stabilization of the enzyme persulfide and perselenide intermediates during catalysis, a necessary intermediate in the formation of sulfur and selenium containing metabolites.     

Specific occurrence of selenium in enzymes and amino acid tRNAs

In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes. Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources. In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center. It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins. The factors that allow this unusual use of the stop codon are, however, unknown. The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established. The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes. The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium.     

Selenopeptide chemistry

This review focuses on the chemical aspects of the 21st proteinogenic amino acid, selenocysteine in peptides and proteins. It describes the physicochemical properties of selenium/sulfur and selenocysteine/cysteine based on comprehensive structural (X‐ray, NMR, CD) and biological data, and illustrates why selenocysteine is considered the most conservative substitution of cysteine. The main focus lies on the synthetic methods on selenocysteine incorporation into peptides and proteins, including an overview of the selenocysteine building block syntheses for Boc‐ and Fmoc‐SPPS. Selenocysteine‐mediated reactions such as native chemical ligation and dehydroalanine formation are addressed towards peptide conjugation. Selenopeptides have very interesting and distinct properties which lead to a diverse range of applications such as structural, functional and mechanistic probes, robust scaffolds, enzymatic reaction design, peptide conjugations and folding tools. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.     

Expression of a mouse selenocysteine lyase in Brassica juncea chloroplasts affects selenium tolerance and accumulation

Selenium is an essential nutrient for many organisms, as part of certain selenoproteins. However, selenium is toxic at high levels, which is thought to be due to non-specific replacement of cysteine by selenocysteine leading to disruption of protein function. In an attempt to prevent non-specific incorporation of selenocysteine into proteins and to possibly enhance plant selenium tolerance and accumulation, a mouse selenocysteine lyase was expressed in Brassica juncea (Indian mustard) chloroplasts, the site of selenocysteine synthesis. This selenocysteine lyase specifically breaks down selenocysteine into elemental selenium and alanine. The transgenic cpSL plants showed normal growth under standard conditions. Selenocysteine lyase activity in the cpSL transgenics was up to 6-fold higher than in wild-type plants. The cpSL transgenics contained up to 40% less selenium in protein compared to wild-type plants, indicating that Se flow in the plant was successfully redirected. Surprisingly, the selenium tolerance of the transgenic cpSL plants was reduced, perhaps due to interference of produced elemental selenium with chloroplastic sulphur metabolism. Shoot selenium levels were enhanced up to 50% in the cpSL transgenics, but only during the seedling stage.     

硒蛋白合成的特殊机制

硒蛋白含有一种特殊氨基酸--硒代半胱氨酸。在翻译阶段,该氨基酸从硒蛋白ｍＲＮＡ编码区的ＵＧＡ密码子处掺入多肽链。已证明它由丝氨酸和活性硒供体分子合成。一种独特的ｔＲＮＡ、某些特殊蛋白质因子以及硒蛋白ｍＲＮＡ的特殊二级结构是ＵＧＡ解读为硒代半胱氨酸所必需的。     

Biochemical discrimination between selenium and sulfur 1: a single residue provides selenium specificity to human selenocysteine lyase

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.     

Selenocysteine: the 21st amino acid

Great excitement was elicited in the field of selenium biochemistry in 1986 by the parallel discoveries that the genes encoding the selenoproteins glutathione peroxidase and bacterial formate dehydrogenase each contain an in-frame TGA codon within their coding sequence. We now know that this codon directs the incorporation of selenium, in the form of selenocysteine, into these proteins. Working with the bacterial system has led to a rapid increase in our knowledge of selenocysteine biosynthesis and to the exciting discovery that this system can now be regarded as an expansion of the genetic code. The prerequisites for such a definition are co-translational insertion into the polypeptide chain and the occurrence of a tRNA molecule which carries selenocysteine. Both of these criteria are fulfilled and, moreover, tRNASec even has its own special translation factor which delivers it to the translating ribosome. It is the aim of this article to review the events leading to the elucidation of selenocysteine as being the 21st amino acid.     

Assessing the dispersive and electrostatic components of the selenium–aromatic interaction energy by DFT

Selenium has been increasingly recognized as an important element in biological systems, which participates in numerous biochemical processes in organisms, notably in enzyme reactions. Selenium can substitute sulfur of cysteine and methionine to form their selenium analogues, selenocysteine (Sec) and selenomethionine (SeM). The nature of amino acid pockets in proteins is dependent on their composition and thus different non-covalent forces determine the interactions between selenium of Sec or SeM and other functional groups, resulting in specific biophysical behavior. The discrimination of selenium toward sulfur has been reported. In order to elucidate the difference between the nature of S-π and Se-π interactions, we performed extensive DFT calculations of dispersive and electrostatic contributions of Se-π interactions in substituted benzenes/hydrogen selenide (H₂Se) complexes. The results are compared with our earlier reported S-π calculations, as well as with available experimental data. Our results show a larger contribution of dispersive interactions in Se-π systems than in S-π ones, which mainly originate from the attraction between Se and substituent groups. We found that selenium exhibits a strong interaction with aromatic systems and may thus play a significant role in stabilizing protein folds and protein–inhibitor complexes. Our findings can also provide molecular insights for understanding enzymatic specificity discrimination between single selenium versus a sulfur atom, notwithstanding their very similar chemical properties.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.     

Isosteric replacement of sulfur with other chalcogens in peptides and proteins.

The review addresses the functional and structural properties of the two series of chalcogen analogues of amino acids in peptides and proteins, the methionine and the serine/cysteine series, and discusses the synthesis of the related selenium/tellurium analogues as well as their use in peptide synthesis and protein expression. Advances in synthetic methodologies and recombinant technologies and their combined applications in native and expressed protein ligation allows the isomorphous character of selenium- and tellurium-containing amino acids to be exploited for production of heavy metal mutants of proteins and thus to facilitate the phasing problem in x-ray crystallography. In addition, selenocysteine has been recognized as an ideal tool for the production of selenoenzymes with new catalytic activities. Moreover, the fully isomorphous character of disulfide replacement with diselenide is well suited to increase the robustness of cystine frameworks in cystine-rich peptides and proteins and for the de novo design of even non-native cystine frameworks by exploiting the highly negative redox potential of selenols.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.     

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP. Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively. Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis.     

A bioassay based on recombinant DNA technology for determining selenium concentration.

The trace element selenium has recently attracted attention, particularly because (i) selenocysteine is involved in the active site of various prokaryotic and eukaryotic enzymes, some of which have a role in human health; (ii) selenocysteine incorporation into these proteins is coded by UGA codons; and (iii) as a result, selenocysteine is now considered to be the 21st amino acid in an expanded genetic code. Here, we built recombinant DNA constructs in which expression of the lac'Z gene is driven in Escherichia coli by UGA-directed selenocysteine incorporation. In this system, levels of beta-galactosidase activity are proportionally and specifically related to the presence and concentrations of several specific simple selenium derivatives. The system can thus be used as a sensitive bioassay for their determination. This bioassay is one of a few using recombinant DNA technology to provide a reporter for simple detection of a chemical trace element.     

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.