ANNUAL REVIEWS
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Quick links to online content Annu. Rev. Biochem. 1990. 59:lJ 1-27
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SELENIUM BIOCHEMISTRY! Thressa C. Stadtman
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 KEY WORDS:
selenium-dependent enzymes, seleno-tRNAs, selenocysteine. UGA codon, selenocystyl-tRNA.
CONTENTS ...... ............. . . . .... . . . ..... . .... . . ..... ".
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SELENOENZYMES THAT CONTAIN SELENOCYSTEINE ............................... Formate Dehydmgenases.......... .... .............. ........................................... Glycine Reductase Selenoprotein A ..... .............. ........................................ Hydrogenases...................................................................................... Mammalian Glutathione Pero.xidases .... . . ... .......................... ......................
113 113 114 115 116
OTHER MAMMALIAN SELENOPROTEINS ..................................................
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PERSPECTIVES AND SUMMARY .. .
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BACTERIAL SELENOENZYMES THAT DO NOT CONTAIN SELENOCYSTEINE ....................................................................
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SELENO-tRNAs..... ........ ............. .................... .................... ................ .....
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MECHANISM OF SPECIFIC INCORPORATION OF SELENOCYSTEINE INTO PROTEINS................................................................................ Selenoprotein Genes That Contain the TGA Codon ... ...................... .............. E. coli and S. typhimurium Genes Affecting Selenium Metabolism . ... . ... . tRNAs with Anticodons Complementary to UGA .................. ......................... The selB Gene Product is a New GTP-binding Translation Factor ............. ....... Role of the selD Gene Product ........................................... .............. . ......
119 119 120 121 122 122
RELATIVE EFFECTIVENESS OF SELENIUM VERSUS SULFUR IN FORMATE DEHYDROGENASE .... . . . . . . . . . ... . . . . ..... . . .. . . . ... .. ...............
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CHEMICAL SYNTHESIS OF SELENOCYSTEINE-CONTAINING ENZYMES ...... .
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ENZYMES THAT DEGRADE SELENOCYSTEINE AND SELENOCYSTINE.........
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DETOXIFICATION OF SELENIUM COMPOUNDS BY METHyLATION.............
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PERSPECTIVES AND SUMMARY The increasing emphasis on selenium-oriented research is evidenced by the ever larger number of attendees at international symposia devoted to chemi cal, biochemical, and medical aspects of selenium as well as smaller sym posia organized within other types of meetings. The present review is devoted primarily to developments in the field of selenium biochemistry that have occurred since 1980. For background information and discussion of research findings prior to 1980, the reader is referred to earlier reviews by this author (1-3). One of the striking new developments in the field of selenium biochemistry stems from the finding that the UGA termination codon is used to direct the covalent insertion of selenocysteine into certain selenium-dependent enzymes (4, 5). Thus, utilization of a particular codon to specify the insertion of the selenoamino acid itself rather than its incorporation by a posttranslational modification mechanism indicates that selenocysteine can be viewed as the 21st amino acid in terms of ribosome-mediated protein synthesis. A puzzling and as-yet-unanswered question has as its focus the factors that are involved in this unusual usage of the UGA termination codon. Specifically, what prevents insertion of selenocysteine in response to UGA at the end of a message yet allows it to be read as selenocysteine when the UGA codon is "in frame"? These and other aspects of the problem will be discussed in sections that describe advances based on use of the genetic approach and, to some extent, classical enzymological methods. Recent studies on mammalian selenoproteins have elucidated interesting properties of a plasma protein, termed protein P, of as-yet-unidentified func tion that accounts for a significant portion of the selenium content of rat plasma (6). A form of selenoglutathione peroxidase also in plasma may account for most of the remaining selenium that is specifically incorporated in proteins. In addition to the well-documented role of glutathione peroxidase as a scavenger of various types of organic peroxides, its possible function in the arachidonic acid cascade of reactions leading to synthesis of prostaglandins has been investigated further. In particular, the specific roles of the selenium dependent glutathione peroxidase and the nonselenium enzyme (glutathione S-transferase) in the process have been studied in an in vitro system (7). In several studies on the anticarcinogenic properties of selenium, a number of proteins radiolabeled with 75Se have been detected in livers of the treated animals. Isolation and partial characterization of a low-molecular-weight species from mouse liver indicated its possible identity with liver fatty acid-binding protein (8). Dissociation of the bound selenium from this protein by treatment with aqueous ethanol at acidic or basic pH values suggests its presence as a noncovalent species and reemphasizes the need to characterize
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rigorously the nature of the binding of selenium to unknown proteins before characterizing them as specific selenoproteins. Selenium toxicity studies prompted by deleterious effects of high levels of the element on migratory birds nesting in irrigation water reservoirs in certain western states in the United States pinpoint problems in developmental embryology that should be investigated. Attempts to convert the high levels of selenite and selenate present in the water to less toxic elemental selenium using specialized bacterial species show some promise as a method of treat ment. Considerable attention to a variety of methylation reactions that de toxify various selenium compounds by conversion to dimethyl selenide and trimethylselenonium ion increased understanding of these reduction and methylation processes (9, 10) .
SELENOENZYMES THAT CONTAIN SELENOCYSTEINE Three types of redox enzymes that contain selenium in the form of selenocys teine have been isolated and characterized from bacterial sources, whereas only one, glutathione peroxidase, is known in mammals. Rigorous proof that selenocysteine-containing glutathione peroxidases also are present in other eukaryotes, such as insects and yeasts, appears to be lacking. Formate Dehydrogenases The initial discovery (11) that selenium is required for the expression of an enzyme activity was made during investigations of the trace elements needed for formate dehydrogenase synthesis by Escherichia coli. Two distinct for mate dehydrogenases are elaborated by E. coli and also by Salmonella species. One of these, a high-molecular-weight complex consisting of three different types of subunits (12), is expressed under anaerobic conditions when nitrate is present as electron acceptor and is linked to nitrate reductase by ubiquinone (12). The other formate dehydrogenase, part of a complex known as formate-hydrogen lyase (13), is expressed under anaerobic conditions in the absence of other added electron acceptors. This enzyme is linked to a hydrogenase, and molecular hydrogen is evolved. The 11O,OOO-dalton selenium-containing subunits of the nitrate reductase-linked formate de hydrogenase and the gO,OOO-dalton selenocysteine-containing subunit of the formate-hydrogen lyase complex are products of two separate genes (14, 15). Although the nitrate reductase-linked enzyme was isolated and characterized more than 10 years ago (12), the genes encoding the enzyme subunits have not been isolated. In contrast, detailed genetic studies of the hydrogenase linked formate dehydrogenase (see later sections) preceded isolation and characterization of the enzyme, which has been accomplished only recently ( M. J. Axley, unpublished). The marked oxygen sensitivity of the selenium-
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containing bacterial fonnate dehydrogenases, as well as several of the non selenium dependent fonnate dehydrogenases of anaerobic bacteria, has con tributed to the relatively slow progress in characterizing these enzymes. In addition, the tendency to lose variable amounts of molybdopterin cofactor and iron sulfur centers during isolation of these enzymes, together with the problems due to the hydrophobic properties of some, have been contributing factors. Fonnate dehydrogenases present in various methane-producing bacteria that utilize fonnate as a sole fennentable substrate differ considerably in properties. One type, purified from Methanococcus vannielii. is a 105,000dalton protein that contains molybdenum and iron sulfur centers but no selenium ( 16). This protein is the major fonnate dehydrogenase species when the organism is cultured in selenium-deficient media. Under these conditions, growth of the organism is very slow. When selenium is supplied (1 J,LM), the organism grows rapidly, and a large fonnate dehydrogenase complex consist ing of selenocysteine-containing subunits of approximately 100,000 daltons ( 16, 17) and the lOS,OOO-dalton molybdenum-iron sulfur-containing subunits is the major fonn of the enzyme present in the cells. It is suggested that the selenopolypeptide-containing enzyme complex is the more active catalytic species. Another methane organism, Methanobacterium formicicum, lacks the ability to produce a selenium-containing fonnate dehydrogenase and instead contains only a molybdopterin-iron sulfur type of catalyst ( 18, 19). High concentrations of this protein in M. formicicum cells may compensate for the lack of selenium ( see later). A selenium-dependent fonnate de hydrogenase in which molybdenum is largely replaced with tungsten occurs in Clostridium thermoaceticum (20). This enzyme uses NADP+ as electron acceptor and in this respect differs from the fonnate dehydrogenases that occur in several of the methane bacteria and E. coli.
Glycine Reductase Selenoprotein A The selenium-dependent glycine reductase complex that is present in a num ber of amino acid-fennenting clostridia contains a small, acidic, heat-stable selenoprotein, tenned selenoprotein A (21). Identification of the selenium containing moiety present in this protein as the amino acid, selenocysteine, provided the first example of its specific occurrence in a protein (22, 23). In addition to selenocysteine, there are two cysteine residues in selenoprotein A, and in the reduced fonn of the protein all three groups are titratable with sulfhydryl reagents and all three react rapidly with oxygen. The oxidized fonn of the protein, isolated as a dimer, can be converted to the biologically active fonn by treatment with dithiolthreitol or borohydride. In the in vitro glycine reductase system, dithiolthreitol is employed as electron donor for the reduc tion of glycine to acetate and ammonia and thus the selenoprotein probably
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serves as the immediate reductant in the react.ion. The partial amino acid sequence of selenoprotein A in the region surrounding the selenocysteine residue has been determined (24) and is as follows: -Cys-Phe-Val-Secys-Thr Ala-Ala-Gly-Ala-Met-Asp-Leu-Glu-Asn-Gln-Lys-. Due to a blocked amino terminus, the amino acid sequence of the amino terminal portion of the protein isolated from Clostridium sticklandii has not been determined. More extensive sequence data have been obtained for another form of selenoprotein A that is not N-blocked. This protein, isolated from Clostridium purinolyti cum, has an identical amino acid sequence in the selenocysteine portion of the molecule ( G. Garcia, unpublished). These and other peptide sequence data may allow generation of ribonucleotide probes sufficiently specific to allow isolation of a gene encoding selenoprotein A. Although the proteins from the two microorganisms are not completely interchangeable as components of the two glycine reductase systems and are not identical in amino acid composi tion, they are similar functionally and immunologically (25, 26). From the protein chemistry standpoint, the small size, the stability, and the ease of isolation of selenoprotein A make it an ideal biological material for studies on the mechanism of selenoprotein synthesis. Preliminary NMR studies on [77 SeJ selenoprotein A isolated from cells of C. sticklandii grown in media contain ing 77Se03- indicate 77Se NMR to be a promising approach for detailed characterization of this selenoprotein (R. S. Glass, and T. C. Stadtman, unpublished) . Hydrogenases
The first hydrogenase that was shown to contain selenium was isolated from
M. vannielii (27,28). This enzyme, Mr 340,000, contains four selenocysteine residues, which are located in four 42,OOO-dalton subunits. Two other types of subunits, two 56,000-dalton a-subunits, which are actually dimers of 27,000 daltons, and two 35,000-dalton ,},-subunits, are present in the com ple,x. Two gram atoms of zinc, 18-20 FeS groups, and two equivalents of FAD are bound to the enzyme. There is evidence that the ionized selenol serves as one of the redox centers of the enzyme. A hydrogenase of similar size and composition has been isolated from Methanococcus voltae (29). Hydrogenases from strains of Methanoautotrophicum, although similar in subunit composition, apparently belong to the group of NiFe hydrogenases that do not contain selenium ( 30, 31). A hydrogenase from Desulfovibrio baculatus, an enzyme of the NiFeSe group, is presumed to contain selenocys teine based on the finding of a TGA codon in the gene encoding the enzyme (31, 32). Broadening of the Ni EPR signal of the reduced protein when the enzyme was prepared from cells grown in the presence of 77Se suggested selenium coordination to Ni in this hydrogenase.
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A hydrogenase from Bradyrhizobium japonicum, a nitrogen-fixing symbi otic organism that forms nodules on the roots of higher plants, copurified with 75Se, but the presence of selenium in each of the two nonidentical subunits with a total of one equivalent or less in the protein dimer suggests the nonspecific incorporation of selenomethionine as an explanation of the observations (33). Furthermore, a TGA codon was not found when the gene encoding this hydrogenase was sequenced (34). Since, from the gene se quence, one �62,000 Mr subunit contains 10 and the other 35 000 Mr subunit contains 11 methionine residues, it seems likely that random, nonspecific incorporation of an occasional selenomethionine instead of methionine could account for the amount of selenium detected in the protein. However, the reported stimulation of growth of the organism and increased specific activity of the hydrogenase in response to added selenium in the growth medium suggest that there may be an alternative explanation.
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�
,
Mammalian Glutathione Peroxidases
The tetrameric glutathione peroxidases found in most mammalian tissues are the best characterized forms of this enzyme. These enzymes contain one selenocysteine residue in each of the four identical subunits, and the ionized selenol moiety participates as the redox center in the peroxidase. A variety of organic peroxides and H202 are decomposed by the enzyme. The structure and function of this enzyme are described in numerous reviews (35, 36). Another glutathione peroxidase, also a tetrameric form, occurs in plasma as a glycosylated protein (37, 38). The subunit of this glycoprotein migrates as a slightly larger species than the subunit of erythrocyte glutathione peroxidase when subjected to electrophoresis in SDS polyacrylamide gels. The plasma enzyme also appears to differ from the erythrocyte peroxidase based on kinetic behavior and immunological properties (38). A different glutathione peroxidase, a monomeric form of 21,000 daltons, is a highly hydrophobic protein that contains selenocysteine and preferentially reduces membrane bound hydroperoxides (39, 40). The enzyme has been isolated from liver and is present in numerous tissues. It is referred to as "phospholipid hydroperox ide glutathione peroxidase." An anti-inflammatory drug known as ebselen or PZ51 (41) exhibits glu tathi�ne peroxidase-like properties in vitro (42) and has been investigated in
/(5" 8--;' �s)�
Scheme
1
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(42, 43). In the reduced form compound the heterocyclic ring is opened to give the selenol (-SeH) (Scheme I). mechanistic studies as a model for the enzyme of the
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OTHER MAMMALIAN SELENOPROTEINS A protein of unknown function in rat plasma termed selenoprotein P (44, 45) accounts for about two-thirds of the selenium in the plasma (R. F. Burk, personal communication), and the glycosylated form of glutathione per oxidase accounts for most of the remaining specifically incorporated sele nium. Selenoprotein P was purified to homogeneity from rat plasma using a
monoclonal antibody affinity matrix (46). This protein, also a glycoprotein, migrated as a single band with an estimated molecular weight of 57,000 when subjected to SDS polyacrylamide gel electrophoretic analysis. Using a radioimmunoassay, the amount of selenoprotein P in normal rat plasma was estimated to be about 50 p,g per ml (47). Less than one-tenth of this amount was found in the plasma of selenium-deficient rats. Following injection of 50 f-tg of selenium as selenite into the selenium-deficient rats, the level of
selenoprotein P reached 52% of control in 6 hours and 7 8% of control in 12 hours (6). In contrast, the glutathione peroxidase level had risen to only 5% of control in 12 hours. Administration of graded doses of selenate in the diet to selenium-deficient rats followed by measurement of selenoprotein P and glutathione peroxidase levels in plasma indicated that the selenium require ment for normal selenoprotein P synthesis is saturated at about one-fifth the concentration required for serum glutathione peroxidase levels to return to normal (6, 47). Preliminary compositional analyses on selenoprotein P indi cate the presence of several selenocysteine residues and a high content of cysteine and histidine (R. F. Burk, personal communication). The biological function of a protein of this unusual composition is the subject of considerable interest.
In studies on the administration of selenium as a chemopreventative agent, BALB/c
a number of proteins labeled with 7SSe have been isolated from
mouse tissues (48). The partial amino acid sequence of one of these, a
14,000-dalton protein, revealed a high degree of homology with rat liver fatty acid binding protein (8). Based on this knowledge, a purification procedure using an oleic acid affinity matrix was designed. Radioactive selenium re mained bound to the protein during elution from the affinity matrix with
30-50% aqueous ethanol, p H 6, but if acidic (pH 2.4) 50% ethanol or 50% ethanol containing 25 mM NaOH was used as eluant, none of the 75Se remained with the protein. From these properties, it is clear that selenium was associated with the protein in an easily dissociable form and was not present as selenocysteine or selenomethionine in the polypeptide.
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BACTERIAL SELENOENZYMES THAT DO NOT CONTAIN SELENOCYSTEINE Earlier reports that formation of two bacterial enzymes, nicotinic acid hydrox ylase (49) and clostridial xanthine dehydrogenase (50), is stimulated by supplementary selenium in the growth medium have been extended to show the copurification of 7SSe and enzyme activity during isolation of the proteins (51, 52; T. C. Stadtman, unpublished). The instantaneous inhibition of
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enzyme activity upon addition of an alkylating agent to reaction mixtures containing nicotinic acid hydroxylase is consistent with the involvement of a very reactive ionized selenol in the catalytic reaction (51). However, the stable alkyl derivative isolated proved in every instance to be a dialkyl selenide. Furthermore, treatment of the native enzyme with various chaotro pic reagents and heat or heating alone released a labile selenium compound from the protein. When prepared under strictly anaerobic conditions and separated from the protein by gel filtration on a Sephadex G-25 column, the selenium compound and the molybdopterin cofactor present in the enzyme were both recovered in a fraction that bound to the Sephadex matrix (52). Although the properties determined so far are not sufficient to identify the selenium moiety of nicotinic acid hydroxylase, they do show that it is not selenocysteine or selenomethionine. Selenoproteins isolated from a fatty acid-producing anaerobic organism,
Clostridium kluyveri, and identified as thiolase and f3-hydroxybutyryl-CoA dehydrogenase were conspicuously labeled with 75Se when the organism was grown in the presence of 1 J.LM or less of CSSe]selenite (53,54). The selenium moiety in the proteins was identified as selenomethionine and the selenoami no acid was distributed randomly throughout the polypeptides in a manner that paralleled methionine (53, 55).
Since both enzymes are rich in
methionine and are present in high concentrations in the cell, the amount of selenomethionine incorporated, even at normal levels of selenium, is con spicuous when radiolabeled selenium is employed for detection. Other en zymes rich in methionine such as f3-galactosidase and glutamine synthetase, also can become significantly labeled with radioactive selenomethionine when cells are grown in media containing J.LM levels of 7sSe-labeled com pounds. The common practice of using the ,a-galactosidase gene as a reporter in gene constructs is the source of additional problems when 7SSe incorpora tion is to be monitored, because of the background of radioactive sele nomethionine that results.
SELENO-tRNAS The specific occurrence of selenium in another class of biopolymers, the amino acid transfer ribonucleic acids, has been documented in several an-
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aerobic bacteria including C. sticklandii (56, 57) and M. vannielii (58), in E. coli (59) and Salmonella ty phimurium (60), in a murine leukemia cell line (61), and in cultured cells of higher plants (62). Glutamate and lysine isoaccepting tRNAs are the species that contain the most conspicuous amounts of selenium in the bacteria that have been examined. In these tRNAs the selenium is present in 5-methylaminomethyl-2-selenouridine residues in the "wobble position" of the anticodons (63, 64). The incorporation of selenium into this nucleoside occurs by an ATP-dependent process that involves n:placement of sulfur from the corresponding thionucleoside, 5methylaminomethyl-2-thiouridine in the tRNA (65). Incorporation of sele nium in this process involves the product of the seLD gene (see later). The presence of selenium in the "wobble position" uridines instead of sulfur seems to affect codon-anticodon interaction. Whereas lysine and glutamate codons ending in A are generally favored when sulfur is present, this bias appears to be canceled by selenium substitution (66).
MECHANISM OF SPECIFIC INCORPORATION OF SELENOCYSTEINE INTO PROTEINS The discovery that a UGA termination (opal) codon is used to direct the cotranslational incorporation of selenocysteine into certain prokaryotic and eukaryotic selenoproteins has extended the selenium research field to include a number of molecular biology specialists. It is already evident that usage of a codon, normally specifying termination, to also direct the cotranslational insertion of selenocysteine requires a complex system made up of previously undetected factors. Unique seryl-tRNAs with anticodons complementary to UGA, enzymes required for the conversion of seryl-tRNA to selenocystyl tRNA, a new elongation factor, and other proteins not yet characterized are among the components of the system detected to date. Additionally, there is evidence that structural constraints in the RNA message itself in the neighbor hood of the UGA codon also are involved. Selenoprotein Genes That Contain the TGA Codon The gene e:ncoding a murine glutathione peroxidase (4) and an E. coli gene, fdhF, encoding the selenopolypeptide of formate dehydrogenase (5) were the first to be shown to contain an in-frame TGA codon. In the case of glutathione peroxidase, the complete amino acid sequence already had been determined for the bovine erythrocyte enzyme (67), and comparison of the de oxyribonucleotide and amino acid sequence data showed that the TGA codon corresponds to the position of the selenocysteine residue in the protein subunit. The E. coli formate dehydrogenase, which is a component of a complex known as formate hydrogen lyase, had never been isolated and thus no amino acid sequence data were available for comparison. However, by
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fusing N-terminal portions of the formate dehydrogenase selenopolypeptide gene to the ,a-galactosidase gene, it could be shown that when constructs included the TGA codon upstream from the ,a-galactosidase gene, selenium was required for readthrough of the message and synthesis of ,a-galactosidase. If the TGA codon was not present in the fusion gene, ,a-galactosidase expression was independent of selenium. Similarly, changing the TGA to TGC or TGT (cysteine codons) or TCA (serine) eliminated the selenium requirement for readthrough of the message (68). When thefdhF gene product was formed in a T7 expression system, a short polypeptide corresponding in size to that expected for the portion from the N-terminus to the TGA position resulted (69). However, no termination occurred when mutated jdhF genes containing TGC, TGT, or TCA instead of TGA were expressed. Thus, in a system wherein translation is limited by selenocysteine availability, termina tion occurs at the UGA codon. From these and similar data, it was clear that UGA in the message must correspond to selenocysteine in the selenopolypep tide even though amino acid sequence data were lacking. Recently, other glutathione peroxidase gene sequences have been determined, and all are highly conserved. The TGA codon was found in the expected position in human (70-72) and rat (73) glutathione peroxidase genes. Selenium-containing hydrogenases have been isolated from various sulfate reducing anaerobic bacteria, and the gene encoding the hydrogenase from D. bacu tatu s was cloned and sequenced (74, 32). A TGA codon detected near the end of the opcn reading frame of this gene indicates that the protein should contain selenocysteine, but this has not been isolated and identified. Howev er, based on the previously demonstrated occurrence of selenocysteine in a hydrogenase isolated from a methane-producing anaerobic organism, the assumption probably is correct.
E. coli and S. typhimurium Genes Affecting Selenium Metabolism A collection of E. coli and S. typhimurium mutants that were selected on the basis of inability to synthesize selenium-dependent formate dehydrogenases and seleno-tRNAs have proven invaluable in elucidating essential steps in the selenium incorporation processes. Whereas some of the mutations prevented the incorporation of selenium into the selenopolypeptides of the formate dehydrogenases but had no effect on selenation of tRNAs, a lesion in one gene resulted in the loss of ability to insert selenium in both types of macromolecules. These mutants, originally assigned to genetic classes desig natedfdhA, fdhB, andfdhC (75), or selAl (60), have been reclassified and the genes are now referred to as setA, setB, setc, and seLD (69). The setA, setB, and seLD gene products are proteins and the selC gene product is a new tRNA (69, 76-78). Although no mutants have been reported that lack the
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ability to convert 2-thiouridine residues of tRNAs to 2-selenouridines but still retain the ability to insert selenium into formate dehydrogenase selenopoly peptides, an asuE mutant that lacks the 2-thiouridines (79) also lacks 2selenouridines in tRNAs (60). Since this strain maintained ability to in corporate selenium into formate dehydrogenase, it was concluded that seleno tRNA does not serve as a source of selenium for selenoprotein synthesis (60). Furthermore, the precursor role of 2-thiouridine residues in tRNAs for seleno tRNA synthesis is supported. tRNAs With Anticodons Complementary to UGA A new tRNA discovered in E. coli that is the product of the selC gene (76) and a mammalian UGA suppressor tRNA formerly designated as phosphoseryl tRNA (80, 81) have anticodons that recognize the UGA codon and are aminoacylated with L-serine by seryl-tRNA ligase. The esterified L-serine residues are phosphorylated by specific kinases (82, 83), and the resulting o-phosphoseryl-tRNAs are then converted to selenocystyl-tRNAs (84, 85) (Scheme 2). Thus, in these cases a performed selenocysteine is not esterified directly, but rather it is generated in situ from a serine residue initially esterified to the tRNA. In E. coli two gene products that are required for this process have been identified (69, 77, 84). One of these, the selA gene product, is a 50,OOO-dalton protein that may be the kinase, and the other is the seLD gene product, a 37,OOO-dalton protein presumably functioning in the selenium substitution step. The in vivo formation of selenocystyl-tRNA has been demonstrated both in E. coli (84) and in cultured rat mammary tumor cells (85). The requirement of a functional selC gene for synthesis of the
:
Generation of Se-Cys-t-RNA
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OH P O·C HC-CH20H NH,
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selenopolypeptides of formate dehydrogenase in E. coli provides evidence that the role of the new tRNA is to deliver selenocysteine to the UGA site on the message for incorporation into the growing polypeptide chain. Presum ably the corresponding mammalian tRNA has the same function. The anticodons of the E. coli (76, 77) and the eukaryotic tRNAs (77, 86, 87), as deduced from the gene sequences, are UCA, but in some mammalian tRNAs the uridine in the "wobble position" of the anticodons is modified posttranscriptionally to a 2' -o-methylcytidine or to an unidentified uridine derivative (80). The 2' -o-methylcytidine containing tRNA appears to be the species that is active in selenocystyl-tRNA formation (D. Hatfield, personal communication).
The selB Gene Product is a New GTP-Binding Translation Factor The seLB gene product, a 68,000-dalton protein, has been shown to bind guanine nucleotides and to interact specifically with selenocystyl-tRNASer but not with seryl-tRNASer (78). Thus, the probable role of this protein is to serve as an elongation factor specific for selenocysteine. As one measure of com plex formation between the SELB protein and selenocystyl-tRNASer, the ability to protect against deesterification by alkaline hydrolysis or against tRNA cleavage by ribonuclease was tested. In both assays the SELB protein contributed significantly to the stability of the aminoacylated derivative and to the tRNA itself when the tRNA was in the form of selenocystyl-tRNAs er, but no protection was afforded when it was tested as seryl-tRNAs er. The addition of guanine nucleotides was not required for the effects observed. A compari son of the binding constants exhibited by the SELB protein and EF-Tu for guanine nucleotides indicated similar values for GTP but a much lower affinity for GDP by the SELB protein. Amino acid sequence homology of the amino-terminal portion of the SELB protein with the G-protein family in dicated the portion of the molecule involved in guanine nucleotide binding.
Role of the selD Gene Product Mutants of S. typhimurium (60) and E. coli (69) unable to incorporate selenium specifically into formate dehydrogenase and tRNAs were shown to be defective in a single gene that is now termed selD (69,88). This gene maps at about 21 minutes on the S. typhimurium chromosome and at about 38 minutes on the E. coli chromosome. The product of this gene is a 37,000dalton protein that is required for the conversion of seryl-tRNASer to selenocystyl-tRNASer (77, 84). Although this fact explains the requirement of the selD gene product for biosynthesis of the formate dehydrogenase seleno polypeptide, it does not account for the role of this gene product in the synthesis of a 2-selenouridine residue in tRNA. Because the generation of a
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reactive sdcnium donor could be the common requirement for the two processes, it is possible that the SELD protein fulfills such a role. Replace ment of the activated serine hydroxyl group and an activated sulfur in 2-thiouridine by selenium could thus be controlled by a single gene product. As discussed later, the conversion of an esterified serine in subtilisin to selenocysteine by reaction with a high concentration of hydrogen selenide at elevated temperature (89), together with the known reactivity of the 2thiocyanate derivative of uridine with hydrogen selenide to form 2selenouridine ( 90), may be models for the enzymatic processes. An entirely unexpected effect of a mutation in the seLD gene was observed in a S. typhimurium mutant, selAJ (60). This organism incorporated selenocysteine into numerous proteins of the cell even though it was unable to synthesize the specific selenopolypeptides of formate dehydrogenase (60, 88). In addition to efficient uptake of selenium and incorporation in proteins as selenocysteine, which accounted for 20% of the selenium added to the culture medium, some selenomethionine was incorporated. Selenium containing proteins of an E. coli seLD mutant accounted for 30% of the selenite (0.5 I-tM) provided in the culture medium. In this organism sele nomethionine was the major selenoamino acid in the proteins, and a lesser amount of selenocysteine was present. From these results it is clear that a mutation in the seLD gene does not affect an early step in the pathway of selenium uptake and metabolism. Furthermore, a pathway of synthesis of the selenoamino acids that is specific for selenium and unrelated to the synthesis of selenocystyl-tRNAser is present in these bacteria. Whether the nonspecific incorporation of selenocysteine in these bacterial proteins, in what appears to be an entirely random fashion, is more widespread than currently believed or is a consequence of the seLD mutation is not presently known.
RELATIVE EFFECTIVENESS OF SELENIUM VERSUS SULFUR IN FORMATE DEHYDROGENASE When the complete E. coli formate dehydrogenasejdhF gene in which TGA had been mutagenized to TGC or TGT was expressed in an E. coli host lacking the selA and selB genes (fdhA) , formate dehydrogenase activity was still detectable in the cells. In contrast, conversion of TGA to TeA ( serine) completely abolished enzyme activity. Replacement of selenocysteine with cysteine in formate dehydrogenase by site-specific mutagenesis was estimated to result in a 75-80% decrease in activity of the enzyme ( M. J. Axley, T. C. Stadtman, and A. Bock, unpublished). These otherwise identical enzymes thus provide biological material for more exact assessment of the advantage of a selenol group that is largely ionized at physiological pH over a sulfhydryl group with its higher pKa in a redox catalyst. However, since both the sulfur
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and the selenium enzymes are highly oxygen labile, preparation of fully active species is difficult. A comparison (77) of the sequences of the fdhF gene of E. coli that encodes a selenocysteine-containing protein and the significantly homologous formate dehydrogenase gene from M. formicicum (19) indicates that substitution of TGC for TGA has occurred in nature. The high concentra tion of the non-selenium-containing enzyme in M. formicicum apparently compensates for the lower activity of a cysteine redox center as compared to a selenocysteine redox center.
CHEMICAL SYNTHESIS OF SELENOCYSTEINE-CONTAINING ENZYMES Two approaches have been used for the construction of selenocysteine containing enzyme catalysts. In one case a 25-amino-acid polypeptide was synthesized by the Merrifield method and selenocysteine residues were in serted directly (91). The protein prepared in this way is the copper metal lothionein of Neurospora crassa which, in the native form, contains seven cysteine residues (92). For the selenoprotein a selenocysteine residue was introduced at each of the seven positions normally occupied by cysteine. The protein containing only cysteine was prepared in the same way. A brief report of metal binding and physical chemical properties of the two types of metal lothioneins indicated many similarities (91). Detailed studies on the relative effectiveness of sulfur versus selenium in a protein designed to transport or detoxify metals should provide interesting data. A different method was used to introduce selenocysteine into the active site of subtilisin, thus converting the protease into an acyltransferase. By adapting a method previously used for conversion of the active site serine of subtilisin to cysteine (93), a new catalyst termed selenolsubtilisin that contained sele nocysteine in place of the serine was produced (89). In this procedure the serine hydroxyl group was selectively activated by reaction with phenyl methanesulfonyl fluoride, and the sulfonylated enzyme was treated with 0.5 M hydrogen selenide in aqueous pH 6.8 buffer for 36 hours at 40°C. Yields of 40-50% were reported for selenoenzyme formation. Studies of the properties of selenolsubtilisin indicated it to be a poor catalyst of amide hydrolysis, whereas reactivity with esters and subsequent transfer of the covalently bound acyl group to water or to amines was greatly accelerated.
ENZYMES THAT DEGRADE SELENOCYSTEINE AND SELENOCYSTINE Two pyridoxal phosphate-dependent enzymes, one termed L-selenocysteine p-Iyase and the other D-selenocystine a,p-Iyase, have been isolated in
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homogeneous fonn and characterized. The enzyme active on L-selenocysteine is widely distributed in mammalian tissues and in bacteria and was isolated from pig liver (94) and from Citrobacter freundii (95). The selenoamino acid is decomposed in the presence of dithiolthreitol to give stoichiometric amounts of hydrogen selenide and L-alanine. When the reaction was carried out anaerobically in the absence of dithiolthreitol, elemental selenium was detected as the product of the J3-elimination reaction. The enzyme from pig liver, Mr 85,000-90,000, is a dimer of 48,000 Mr subunits; the C. freundii enzyme is a monomeric protein of 64,000 Mf' The enzymes also differ in amino acid composition and several physicochemical properties. However, they are similar in their enzymological properties, and both are inactivated by J3-chloro-L-alanine, a mechanism-based inactivator. Although it is suggested that the physiological role of L-selenocysteine-J3-lyase could be detoxification of the selenoamino acid to elemental selenium, there is no clear-cut evidence on this point. The high Km value (about 1 mM) exhibited by the enzyme from both mammalian and bacterial sources for selenocysteine could be consistent with a detoxification role. The pYlidoxal phosphate-dependent o-selenocystine-a,J3-lyase was de tected in certain anaerobic amino acid fennenting bacteria and was isolated from C. sticklandii (96). This enzyme, Mr 74,000, consists of two identical 35,000 Mr subunits. D-cystine and D-cysteine also serve as substrates, but L-amino acids are inert. The enzyme, although resembling D-cysteine de sulfhydrase of E. coli, was distinguished from the latter by showing that there was no cross-reactivity with antibodies elicited to the two pure enzymes. The products fonned from D-selenocystine by this novel enzyme are pyruvate, ammonia, and elemental selenium. The initial elimination product is thought to be the perselenide of D-selenocysteine (R-Se-Se-). Although a reactive perselenide is attractive as a potential selenium donor for selenoprotein and seleno-tRNA biosynthesis, the enzyme has not been detected as a participant in the processes nor has any clue been found as to its biological role.
DETOXIFICATION OF SELENIUM COMPOUNDS BY METHYLATION The role of methylation as a means of detoxification of selenium compounds is well known as dimethylselenide and trimethylselenonium ion are common excretory products (9, 10). Generation of dimethylselenide, following reduc tion of ingested selenite to hydrogen selenide, converts the highly toxic selenium compounds to a volatile, less toxic fonn. This series of reactions also is catalyzed by a number of fungi. Although it has been generally accepted that the methylated derivatives are dead end products, there is newer evidence to show that demethylation processes are important and recycling of selenium from these compounds occurs (97).
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Literature Cited I. Stadtman, T. C. 1974. Science 183:9152. 3. 4.
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27. Yamazaki, S. 1982. l. Bioi. Chern. 257:7926-29 28. Yamazaki, S. 1984. Selenium in Biology and Medicine, 3rd Int. Symp., Beijing, China, ed. G. F. Combs, Jr. , 1. E. Spall holz, O. A. Levander, J. E. Oldfield, pp. 230-35. New York: AVI Press 29. Muth, E., Morschel, E., Klein, A. 1987. Eur. l. Biochem. 169:571-77 30. Fox, J. A., Livingston, D. J., Orme lohnson, W. H., Walsh, C. T. 1987. Biochemistry 26:4219-27 31. Grahame, D. A. 1988. BioFactors I: 279--83 32. Menon, N. K., Peck, H . D. lr., LeGall, J., Pryzbyla, A. E. 1988. l. Bacterial. 170:4429 33. Boursier, P., Hanus, F. 1., Papen, H., Becker, M. M., Russell, S. A., Evans, H.l. 1988. l. Bacterial. 170:5594--600 34. Sayavedra, L. A., Powell, G. K., Evans, H. J. , Morris, R. O. 1989. Proc. Natl. Acad. Sci. USA. In press 35. Flobe, L. 1976. Proc. Symp. Selenium Tellurium, Environmental Industrial Health Foundation, Inc., Pittsburgh, Pa, pp. 138-57 36. Fiohe, L. 1989. Glutathione: Chemical, Biochemical, and Medical Aspects, ed. D. Dolphin, R. Poulson, O. Avamovic, pp. 644-731. Wiley 37. Maddipati, K. R., Marnett, L. J. 1987. l. Bioi. Chem. 262:17398-403 38. Takahashi, K. T., Avissar, N. , Whitin, J., Cohen, H. 1987. Arch. Biochem. Biophys. 256:677-86 39. Ursini, F., Maiorino, M., Valente, M., Gregolin, C. 1982. Biochem. Biophys. Acta 839:62-70 40. Maiorino, M., Roveri, A., Ursini, F. 1988. Phosphorus and Sulfur 38:41-48 41. Kamigata, N. , Iizuka, H., Iyouka, A., Kobayashi, M. 1986. Bull. Chern. Soc. lpn. 59:2179-84 42. Fischer, H., Dereu, N. 1987. Bull. Soc. Chim. Belg. 96:757-63 43. Glass, R. S. , Faroogui, F., Sabahi, M. , Ehler, K. W. 1989. l. Org. Chern. 54: 1092-97 44. Motsenbocker, M. A., Tappel, A. L. 1982. Biochern. Biophys. Acta 704:25360 45. Burk, R. F., Gregory, P. E. 1982. Arch. Biochem. Biophys. 213:73-80 46. Yang, I.-G., Morrison-Plummer, J. , Burk, R. F. 1987. l. Bioi. Chern. 262: 13372-75 47. Yang, J.-G., Hill, K. E., Burk, R. F. 1989. l. Nutr. 119:1010-12 48. Bansal, M. P., Oborn, C. J., Danielson, K. G., Medina, D. 1989. Carcinogene sis 10:541-46
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SELENIUM BIOCHEMISTRY 49. Imhoff. D., Andreesen, J. R. 1979. FEMS Microbial. Lett. 5:155-58 50. Wagner, R., Andreesen, J. R. 1979. Arch. Microbiol. 12 1:255-59 5 1. Dilworth, G. L. 1982. Arch. Biochem. Biophys. 219:30-38 52. Dilworth, G. L. 1983. Arch. Biochem. Biophys. 22 1:565-69 53. Hartmanis, M. G. N., Stadtman, T. C. 1982. Proc. Natl. Acad. Sci. USA 79: 4912-16 54. Sliwkowski. M. X., Hartmanis, M. G. N. 1984. Anal. Biochem. 14 1:344-47 55. Sliwkowski, M. X., Stadtman, T. C. 1985. 1. Bioi. Chem. 260:3140-44 56. Chen, C.-S., Stadtman, T. C. 1980. Proc. NaIl. Acad. Sci. USA 77:1403-7 57. Ching, W.-M., Stadtman, T. C. 1982. Proc. Natl. Acad. Sci. USA 79:37477 58. Ching, W.-M., Wittwer, A. J., Tsai, L., Stadtman, T. C. 1984. Proc. Natl.
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59. Wittwer, A. J. 1 983. J. Bioi. Chem. 258:8637-41 60. Kramer, G. F., Ames, B. N. 1988. J. Bacteriol. 170:736-43 61. Ching, W.-M. 1984. Proc. Natl. Acad. Sci. USA 81:3010-13 62. Wen, T.-N., Li, C., Chen, c.-S. 1988. Plant Sci. 57: 185-93 63. Wittwer, A. J., Tsai, L., Ching, W.-M., Stadtman, 1. C. 1984. Biochemistry 23: 4650-55 64. Ching, W.-M., Alzner-DeWeerd, B., Stadtman, T. C. 1985. Proc. Natl. Acad. Sci. USA 82:347-50 65. Wittwer, A. J., Stadtman, T. C. 1986. Arch. Biochem. Biophys. 248:540-50 66. Wittwer, A. J., Ching, W.-M. 1989. BioFactors 2:27-34 67. Giinzler, W. A., Steffens, G. J., Gross man, A., Kim, S.-M., Otting, F., et al. 1984. Hoppe-Seyler's Z. Physiol. Chem. 365:195-212 68. Zinoni, F., Birkmann, A., Leinfelder, W., Bock, A. 1987. Proc. Nat!. Acad. Sci. USA 84:3 156-60 69. Leinfe1der, W., Forchhammer, K., Zinoni, F., Sawyers, G., Mandrand Berthelot, M.-A., Bock, A. 1988. J. Bacterial. 170:540-46 70. Mullenbach, G. T., Tabrizi, A., Irvine, B. D., Bell, G. I., Hallewell, R. A. 1987. Nucleic Acids Res. 15:5484 7\. Sukenaga, Y., Ishida, K., Takeda, T., Takagi, K. 1987. Nucleic Acids Res. 15:7178 72. Ishida, K., Morino, T., Takagi, K., Sukenaga, Y. 1987. Nucleic Acids Res. 15:10051 73. Reddy, A. P., Hsu, B., Reddy, P. S., Li, N.-Q., Thyagaraju, K., et al. 1988. Nucleic Acids Res. 16:5557-68
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74. Menon, N. K., Peck, H. D. Jr., LeGall, J., Przybyla, A. E. 1987. J. Bacterial. 169:540 1-7 75. Haddock, B. A., Mandrand-Berthelot, M.-A. 1982. Biochem. Soc. Trans. 10: 478-80 76. Leinfelder, W., ZeheIin, E., Mandrand Berthelot, M.-A., Bock, A. 1988. Na ture 331 :723-25 77. Bock, A., Stadtman, T. C. 1988. BioF actors 1 :245-50 78. Forchhammer, K., Leinfelder, W., Bock, A. 1989. Nature. In press 79. Sullivan, M. A., Cannon, J. F., Webb, F. H., Bock, R. M. 1985. J. Bacterial. 161:368-76 80. Diamond, A., Dudock, B., Hatfield, D. 1981. Cell 25:497-506 81. Hatfield, D., Diamond, A., Dudock, B. 1982. Proc. Natl. Acad. Sci. USA 79:62 15-19 82. Mizutani, T., Hashimoto, A. 1984. FEBS Lett. 169:319-22 83. Mizutani, T., Maruyama, N., Hitaka, T., Sukenaga, Y. 1989. FEBS Lett. 247:345-48 84. Leinfelder, W., Stadtman, T. C., Bock, A. 1989. J. Bioi. Chem. 264:9720-23 85. Lee, B. J., Worland, P. J., Davis, 1. N., Stadtman, T. c., Hatfield, D. L. 1989. J. Bioi. Chem. 264:9724-27 86. Lee, B. J., de la Pena, P., Tobian, J., Zasloff, M., Hatfield, D. 1987. Proc. Natl. Acad. Sci. USA 84:6384-88 87. Hatfield, D. 1985. Trends Biachem. Sci. 10:245-50 88. Stadtman, T. C., Davis, J. N., Zehelin, E., Bock. A. 1989. BioFactars 2:35-44 89. Wu. Z-P., Hilvert, D. 1989. J. Am. Chem. Soc. 1 1 1:4513-14 90. Pal, B. C., Schmidt, D. G. 1977.1. Am. Chern. Soc. 99:1973-74 91. Tanaka, H., Oikawa, T., Sugimoto, M., Esaki, N., Soda, K. 1988. 4th Int. Syrnp. Selenium Bioi. Med., Univ. Tiibingen, Tiibingen, West Germany, July 18-21, Abstr. 92. Sugimoto, M., Oikawa, T., Esaki, N., Tanaka, H., Soda, K. 1988. J. Biochem. 104:924-26 93. Polgar, L. 1976. Acta Biochem. Bio phys. Acad. Sci. Hung. 1 1:77-80 94. Esaki, N., Nakamura, T., Tanaka, H., Soda, K. 1982. J. Bioi. Chem. 257: 4386-91 95. Chocat, P., Esaki, N., Tanizawa, K., Nakamura, K., Tanaka, H., Soda, K. 1985. J. Bacterial. 163:669-76 96. Esaki, N., Seraneeprakam, Y., Tanaka, H., Soda, K. 1988. J. Bacterial. 170: 75 1-56 97. Ganther, H. E., Yadhanavikit, S. 1989. 19th Annu. FEBS Meet., Rome, Italy, July 2-7, Abstr. TH80L
Further
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SELENIUM BIOCHEMISTRY! Thressa C. Stadtman
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 KEY WORDS:
selenium-dependent enzymes, seleno-tRNAs, selenocysteine. UGA codon, selenocystyl-tRNA.
CONTENTS ...... ............. . . . .... . . . ..... . .... . . ..... ".
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SELENOENZYMES THAT CONTAIN SELENOCYSTEINE ............................... Formate Dehydmgenases.......... .... .............. ........................................... Glycine Reductase Selenoprotein A ..... .............. ........................................ Hydrogenases...................................................................................... Mammalian Glutathione Pero.xidases .... . . ... .......................... ......................
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OTHER MAMMALIAN SELENOPROTEINS ..................................................
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PERSPECTIVES AND SUMMARY .. .
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BACTERIAL SELENOENZYMES THAT DO NOT CONTAIN SELENOCYSTEINE ....................................................................
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SELENO-tRNAs..... ........ ............. .................... .................... ................ .....
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MECHANISM OF SPECIFIC INCORPORATION OF SELENOCYSTEINE INTO PROTEINS................................................................................ Selenoprotein Genes That Contain the TGA Codon ... ...................... .............. E. coli and S. typhimurium Genes Affecting Selenium Metabolism . ... . ... . tRNAs with Anticodons Complementary to UGA .................. ......................... The selB Gene Product is a New GTP-binding Translation Factor ............. ....... Role of the selD Gene Product ........................................... .............. . ......
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RELATIVE EFFECTIVENESS OF SELENIUM VERSUS SULFUR IN FORMATE DEHYDROGENASE .... . . . . . . . . . ... . . . . ..... . . .. . . . ... .. ...............
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CHEMICAL SYNTHESIS OF SELENOCYSTEINE-CONTAINING ENZYMES ...... .
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ENZYMES THAT DEGRADE SELENOCYSTEINE AND SELENOCYSTINE.........
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DETOXIFICATION OF SELENIUM COMPOUNDS BY METHyLATION.............
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PERSPECTIVES AND SUMMARY The increasing emphasis on selenium-oriented research is evidenced by the ever larger number of attendees at international symposia devoted to chemi cal, biochemical, and medical aspects of selenium as well as smaller sym posia organized within other types of meetings. The present review is devoted primarily to developments in the field of selenium biochemistry that have occurred since 1980. For background information and discussion of research findings prior to 1980, the reader is referred to earlier reviews by this author (1-3). One of the striking new developments in the field of selenium biochemistry stems from the finding that the UGA termination codon is used to direct the covalent insertion of selenocysteine into certain selenium-dependent enzymes (4, 5). Thus, utilization of a particular codon to specify the insertion of the selenoamino acid itself rather than its incorporation by a posttranslational modification mechanism indicates that selenocysteine can be viewed as the 21st amino acid in terms of ribosome-mediated protein synthesis. A puzzling and as-yet-unanswered question has as its focus the factors that are involved in this unusual usage of the UGA termination codon. Specifically, what prevents insertion of selenocysteine in response to UGA at the end of a message yet allows it to be read as selenocysteine when the UGA codon is "in frame"? These and other aspects of the problem will be discussed in sections that describe advances based on use of the genetic approach and, to some extent, classical enzymological methods. Recent studies on mammalian selenoproteins have elucidated interesting properties of a plasma protein, termed protein P, of as-yet-unidentified func tion that accounts for a significant portion of the selenium content of rat plasma (6). A form of selenoglutathione peroxidase also in plasma may account for most of the remaining selenium that is specifically incorporated in proteins. In addition to the well-documented role of glutathione peroxidase as a scavenger of various types of organic peroxides, its possible function in the arachidonic acid cascade of reactions leading to synthesis of prostaglandins has been investigated further. In particular, the specific roles of the selenium dependent glutathione peroxidase and the nonselenium enzyme (glutathione S-transferase) in the process have been studied in an in vitro system (7). In several studies on the anticarcinogenic properties of selenium, a number of proteins radiolabeled with 75Se have been detected in livers of the treated animals. Isolation and partial characterization of a low-molecular-weight species from mouse liver indicated its possible identity with liver fatty acid-binding protein (8). Dissociation of the bound selenium from this protein by treatment with aqueous ethanol at acidic or basic pH values suggests its presence as a noncovalent species and reemphasizes the need to characterize
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rigorously the nature of the binding of selenium to unknown proteins before characterizing them as specific selenoproteins. Selenium toxicity studies prompted by deleterious effects of high levels of the element on migratory birds nesting in irrigation water reservoirs in certain western states in the United States pinpoint problems in developmental embryology that should be investigated. Attempts to convert the high levels of selenite and selenate present in the water to less toxic elemental selenium using specialized bacterial species show some promise as a method of treat ment. Considerable attention to a variety of methylation reactions that de toxify various selenium compounds by conversion to dimethyl selenide and trimethylselenonium ion increased understanding of these reduction and methylation processes (9, 10) .
SELENOENZYMES THAT CONTAIN SELENOCYSTEINE Three types of redox enzymes that contain selenium in the form of selenocys teine have been isolated and characterized from bacterial sources, whereas only one, glutathione peroxidase, is known in mammals. Rigorous proof that selenocysteine-containing glutathione peroxidases also are present in other eukaryotes, such as insects and yeasts, appears to be lacking. Formate Dehydrogenases The initial discovery (11) that selenium is required for the expression of an enzyme activity was made during investigations of the trace elements needed for formate dehydrogenase synthesis by Escherichia coli. Two distinct for mate dehydrogenases are elaborated by E. coli and also by Salmonella species. One of these, a high-molecular-weight complex consisting of three different types of subunits (12), is expressed under anaerobic conditions when nitrate is present as electron acceptor and is linked to nitrate reductase by ubiquinone (12). The other formate dehydrogenase, part of a complex known as formate-hydrogen lyase (13), is expressed under anaerobic conditions in the absence of other added electron acceptors. This enzyme is linked to a hydrogenase, and molecular hydrogen is evolved. The 11O,OOO-dalton selenium-containing subunits of the nitrate reductase-linked formate de hydrogenase and the gO,OOO-dalton selenocysteine-containing subunit of the formate-hydrogen lyase complex are products of two separate genes (14, 15). Although the nitrate reductase-linked enzyme was isolated and characterized more than 10 years ago (12), the genes encoding the enzyme subunits have not been isolated. In contrast, detailed genetic studies of the hydrogenase linked formate dehydrogenase (see later sections) preceded isolation and characterization of the enzyme, which has been accomplished only recently ( M. J. Axley, unpublished). The marked oxygen sensitivity of the selenium-
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containing bacterial fonnate dehydrogenases, as well as several of the non selenium dependent fonnate dehydrogenases of anaerobic bacteria, has con tributed to the relatively slow progress in characterizing these enzymes. In addition, the tendency to lose variable amounts of molybdopterin cofactor and iron sulfur centers during isolation of these enzymes, together with the problems due to the hydrophobic properties of some, have been contributing factors. Fonnate dehydrogenases present in various methane-producing bacteria that utilize fonnate as a sole fennentable substrate differ considerably in properties. One type, purified from Methanococcus vannielii. is a 105,000dalton protein that contains molybdenum and iron sulfur centers but no selenium ( 16). This protein is the major fonnate dehydrogenase species when the organism is cultured in selenium-deficient media. Under these conditions, growth of the organism is very slow. When selenium is supplied (1 J,LM), the organism grows rapidly, and a large fonnate dehydrogenase complex consist ing of selenocysteine-containing subunits of approximately 100,000 daltons ( 16, 17) and the lOS,OOO-dalton molybdenum-iron sulfur-containing subunits is the major fonn of the enzyme present in the cells. It is suggested that the selenopolypeptide-containing enzyme complex is the more active catalytic species. Another methane organism, Methanobacterium formicicum, lacks the ability to produce a selenium-containing fonnate dehydrogenase and instead contains only a molybdopterin-iron sulfur type of catalyst ( 18, 19). High concentrations of this protein in M. formicicum cells may compensate for the lack of selenium ( see later). A selenium-dependent fonnate de hydrogenase in which molybdenum is largely replaced with tungsten occurs in Clostridium thermoaceticum (20). This enzyme uses NADP+ as electron acceptor and in this respect differs from the fonnate dehydrogenases that occur in several of the methane bacteria and E. coli.
Glycine Reductase Selenoprotein A The selenium-dependent glycine reductase complex that is present in a num ber of amino acid-fennenting clostridia contains a small, acidic, heat-stable selenoprotein, tenned selenoprotein A (21). Identification of the selenium containing moiety present in this protein as the amino acid, selenocysteine, provided the first example of its specific occurrence in a protein (22, 23). In addition to selenocysteine, there are two cysteine residues in selenoprotein A, and in the reduced fonn of the protein all three groups are titratable with sulfhydryl reagents and all three react rapidly with oxygen. The oxidized fonn of the protein, isolated as a dimer, can be converted to the biologically active fonn by treatment with dithiolthreitol or borohydride. In the in vitro glycine reductase system, dithiolthreitol is employed as electron donor for the reduc tion of glycine to acetate and ammonia and thus the selenoprotein probably
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serves as the immediate reductant in the react.ion. The partial amino acid sequence of selenoprotein A in the region surrounding the selenocysteine residue has been determined (24) and is as follows: -Cys-Phe-Val-Secys-Thr Ala-Ala-Gly-Ala-Met-Asp-Leu-Glu-Asn-Gln-Lys-. Due to a blocked amino terminus, the amino acid sequence of the amino terminal portion of the protein isolated from Clostridium sticklandii has not been determined. More extensive sequence data have been obtained for another form of selenoprotein A that is not N-blocked. This protein, isolated from Clostridium purinolyti cum, has an identical amino acid sequence in the selenocysteine portion of the molecule ( G. Garcia, unpublished). These and other peptide sequence data may allow generation of ribonucleotide probes sufficiently specific to allow isolation of a gene encoding selenoprotein A. Although the proteins from the two microorganisms are not completely interchangeable as components of the two glycine reductase systems and are not identical in amino acid composi tion, they are similar functionally and immunologically (25, 26). From the protein chemistry standpoint, the small size, the stability, and the ease of isolation of selenoprotein A make it an ideal biological material for studies on the mechanism of selenoprotein synthesis. Preliminary NMR studies on [77 SeJ selenoprotein A isolated from cells of C. sticklandii grown in media contain ing 77Se03- indicate 77Se NMR to be a promising approach for detailed characterization of this selenoprotein (R. S. Glass, and T. C. Stadtman, unpublished) . Hydrogenases
The first hydrogenase that was shown to contain selenium was isolated from
M. vannielii (27,28). This enzyme, Mr 340,000, contains four selenocysteine residues, which are located in four 42,OOO-dalton subunits. Two other types of subunits, two 56,000-dalton a-subunits, which are actually dimers of 27,000 daltons, and two 35,000-dalton ,},-subunits, are present in the com ple,x. Two gram atoms of zinc, 18-20 FeS groups, and two equivalents of FAD are bound to the enzyme. There is evidence that the ionized selenol serves as one of the redox centers of the enzyme. A hydrogenase of similar size and composition has been isolated from Methanococcus voltae (29). Hydrogenases from strains of Methanoautotrophicum, although similar in subunit composition, apparently belong to the group of NiFe hydrogenases that do not contain selenium ( 30, 31). A hydrogenase from Desulfovibrio baculatus, an enzyme of the NiFeSe group, is presumed to contain selenocys teine based on the finding of a TGA codon in the gene encoding the enzyme (31, 32). Broadening of the Ni EPR signal of the reduced protein when the enzyme was prepared from cells grown in the presence of 77Se suggested selenium coordination to Ni in this hydrogenase.
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A hydrogenase from Bradyrhizobium japonicum, a nitrogen-fixing symbi otic organism that forms nodules on the roots of higher plants, copurified with 75Se, but the presence of selenium in each of the two nonidentical subunits with a total of one equivalent or less in the protein dimer suggests the nonspecific incorporation of selenomethionine as an explanation of the observations (33). Furthermore, a TGA codon was not found when the gene encoding this hydrogenase was sequenced (34). Since, from the gene se quence, one �62,000 Mr subunit contains 10 and the other 35 000 Mr subunit contains 11 methionine residues, it seems likely that random, nonspecific incorporation of an occasional selenomethionine instead of methionine could account for the amount of selenium detected in the protein. However, the reported stimulation of growth of the organism and increased specific activity of the hydrogenase in response to added selenium in the growth medium suggest that there may be an alternative explanation.
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�
,
Mammalian Glutathione Peroxidases
The tetrameric glutathione peroxidases found in most mammalian tissues are the best characterized forms of this enzyme. These enzymes contain one selenocysteine residue in each of the four identical subunits, and the ionized selenol moiety participates as the redox center in the peroxidase. A variety of organic peroxides and H202 are decomposed by the enzyme. The structure and function of this enzyme are described in numerous reviews (35, 36). Another glutathione peroxidase, also a tetrameric form, occurs in plasma as a glycosylated protein (37, 38). The subunit of this glycoprotein migrates as a slightly larger species than the subunit of erythrocyte glutathione peroxidase when subjected to electrophoresis in SDS polyacrylamide gels. The plasma enzyme also appears to differ from the erythrocyte peroxidase based on kinetic behavior and immunological properties (38). A different glutathione peroxidase, a monomeric form of 21,000 daltons, is a highly hydrophobic protein that contains selenocysteine and preferentially reduces membrane bound hydroperoxides (39, 40). The enzyme has been isolated from liver and is present in numerous tissues. It is referred to as "phospholipid hydroperox ide glutathione peroxidase." An anti-inflammatory drug known as ebselen or PZ51 (41) exhibits glu tathi�ne peroxidase-like properties in vitro (42) and has been investigated in
/(5" 8--;' �s)�
Scheme
1
SELENIUM BIOCHEMISTRY
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(42, 43). In the reduced form compound the heterocyclic ring is opened to give the selenol (-SeH) (Scheme I). mechanistic studies as a model for the enzyme of the
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OTHER MAMMALIAN SELENOPROTEINS A protein of unknown function in rat plasma termed selenoprotein P (44, 45) accounts for about two-thirds of the selenium in the plasma (R. F. Burk, personal communication), and the glycosylated form of glutathione per oxidase accounts for most of the remaining specifically incorporated sele nium. Selenoprotein P was purified to homogeneity from rat plasma using a
monoclonal antibody affinity matrix (46). This protein, also a glycoprotein, migrated as a single band with an estimated molecular weight of 57,000 when subjected to SDS polyacrylamide gel electrophoretic analysis. Using a radioimmunoassay, the amount of selenoprotein P in normal rat plasma was estimated to be about 50 p,g per ml (47). Less than one-tenth of this amount was found in the plasma of selenium-deficient rats. Following injection of 50 f-tg of selenium as selenite into the selenium-deficient rats, the level of
selenoprotein P reached 52% of control in 6 hours and 7 8% of control in 12 hours (6). In contrast, the glutathione peroxidase level had risen to only 5% of control in 12 hours. Administration of graded doses of selenate in the diet to selenium-deficient rats followed by measurement of selenoprotein P and glutathione peroxidase levels in plasma indicated that the selenium require ment for normal selenoprotein P synthesis is saturated at about one-fifth the concentration required for serum glutathione peroxidase levels to return to normal (6, 47). Preliminary compositional analyses on selenoprotein P indi cate the presence of several selenocysteine residues and a high content of cysteine and histidine (R. F. Burk, personal communication). The biological function of a protein of this unusual composition is the subject of considerable interest.
In studies on the administration of selenium as a chemopreventative agent, BALB/c
a number of proteins labeled with 7SSe have been isolated from
mouse tissues (48). The partial amino acid sequence of one of these, a
14,000-dalton protein, revealed a high degree of homology with rat liver fatty acid binding protein (8). Based on this knowledge, a purification procedure using an oleic acid affinity matrix was designed. Radioactive selenium re mained bound to the protein during elution from the affinity matrix with
30-50% aqueous ethanol, p H 6, but if acidic (pH 2.4) 50% ethanol or 50% ethanol containing 25 mM NaOH was used as eluant, none of the 75Se remained with the protein. From these properties, it is clear that selenium was associated with the protein in an easily dissociable form and was not present as selenocysteine or selenomethionine in the polypeptide.
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BACTERIAL SELENOENZYMES THAT DO NOT CONTAIN SELENOCYSTEINE Earlier reports that formation of two bacterial enzymes, nicotinic acid hydrox ylase (49) and clostridial xanthine dehydrogenase (50), is stimulated by supplementary selenium in the growth medium have been extended to show the copurification of 7SSe and enzyme activity during isolation of the proteins (51, 52; T. C. Stadtman, unpublished). The instantaneous inhibition of
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enzyme activity upon addition of an alkylating agent to reaction mixtures containing nicotinic acid hydroxylase is consistent with the involvement of a very reactive ionized selenol in the catalytic reaction (51). However, the stable alkyl derivative isolated proved in every instance to be a dialkyl selenide. Furthermore, treatment of the native enzyme with various chaotro pic reagents and heat or heating alone released a labile selenium compound from the protein. When prepared under strictly anaerobic conditions and separated from the protein by gel filtration on a Sephadex G-25 column, the selenium compound and the molybdopterin cofactor present in the enzyme were both recovered in a fraction that bound to the Sephadex matrix (52). Although the properties determined so far are not sufficient to identify the selenium moiety of nicotinic acid hydroxylase, they do show that it is not selenocysteine or selenomethionine. Selenoproteins isolated from a fatty acid-producing anaerobic organism,
Clostridium kluyveri, and identified as thiolase and f3-hydroxybutyryl-CoA dehydrogenase were conspicuously labeled with 75Se when the organism was grown in the presence of 1 J.LM or less of CSSe]selenite (53,54). The selenium moiety in the proteins was identified as selenomethionine and the selenoami no acid was distributed randomly throughout the polypeptides in a manner that paralleled methionine (53, 55).
Since both enzymes are rich in
methionine and are present in high concentrations in the cell, the amount of selenomethionine incorporated, even at normal levels of selenium, is con spicuous when radiolabeled selenium is employed for detection. Other en zymes rich in methionine such as f3-galactosidase and glutamine synthetase, also can become significantly labeled with radioactive selenomethionine when cells are grown in media containing J.LM levels of 7sSe-labeled com pounds. The common practice of using the ,a-galactosidase gene as a reporter in gene constructs is the source of additional problems when 7SSe incorpora tion is to be monitored, because of the background of radioactive sele nomethionine that results.
SELENO-tRNAS The specific occurrence of selenium in another class of biopolymers, the amino acid transfer ribonucleic acids, has been documented in several an-
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aerobic bacteria including C. sticklandii (56, 57) and M. vannielii (58), in E. coli (59) and Salmonella ty phimurium (60), in a murine leukemia cell line (61), and in cultured cells of higher plants (62). Glutamate and lysine isoaccepting tRNAs are the species that contain the most conspicuous amounts of selenium in the bacteria that have been examined. In these tRNAs the selenium is present in 5-methylaminomethyl-2-selenouridine residues in the "wobble position" of the anticodons (63, 64). The incorporation of selenium into this nucleoside occurs by an ATP-dependent process that involves n:placement of sulfur from the corresponding thionucleoside, 5methylaminomethyl-2-thiouridine in the tRNA (65). Incorporation of sele nium in this process involves the product of the seLD gene (see later). The presence of selenium in the "wobble position" uridines instead of sulfur seems to affect codon-anticodon interaction. Whereas lysine and glutamate codons ending in A are generally favored when sulfur is present, this bias appears to be canceled by selenium substitution (66).
MECHANISM OF SPECIFIC INCORPORATION OF SELENOCYSTEINE INTO PROTEINS The discovery that a UGA termination (opal) codon is used to direct the cotranslational incorporation of selenocysteine into certain prokaryotic and eukaryotic selenoproteins has extended the selenium research field to include a number of molecular biology specialists. It is already evident that usage of a codon, normally specifying termination, to also direct the cotranslational insertion of selenocysteine requires a complex system made up of previously undetected factors. Unique seryl-tRNAs with anticodons complementary to UGA, enzymes required for the conversion of seryl-tRNA to selenocystyl tRNA, a new elongation factor, and other proteins not yet characterized are among the components of the system detected to date. Additionally, there is evidence that structural constraints in the RNA message itself in the neighbor hood of the UGA codon also are involved. Selenoprotein Genes That Contain the TGA Codon The gene e:ncoding a murine glutathione peroxidase (4) and an E. coli gene, fdhF, encoding the selenopolypeptide of formate dehydrogenase (5) were the first to be shown to contain an in-frame TGA codon. In the case of glutathione peroxidase, the complete amino acid sequence already had been determined for the bovine erythrocyte enzyme (67), and comparison of the de oxyribonucleotide and amino acid sequence data showed that the TGA codon corresponds to the position of the selenocysteine residue in the protein subunit. The E. coli formate dehydrogenase, which is a component of a complex known as formate hydrogen lyase, had never been isolated and thus no amino acid sequence data were available for comparison. However, by
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fusing N-terminal portions of the formate dehydrogenase selenopolypeptide gene to the ,a-galactosidase gene, it could be shown that when constructs included the TGA codon upstream from the ,a-galactosidase gene, selenium was required for readthrough of the message and synthesis of ,a-galactosidase. If the TGA codon was not present in the fusion gene, ,a-galactosidase expression was independent of selenium. Similarly, changing the TGA to TGC or TGT (cysteine codons) or TCA (serine) eliminated the selenium requirement for readthrough of the message (68). When thefdhF gene product was formed in a T7 expression system, a short polypeptide corresponding in size to that expected for the portion from the N-terminus to the TGA position resulted (69). However, no termination occurred when mutated jdhF genes containing TGC, TGT, or TCA instead of TGA were expressed. Thus, in a system wherein translation is limited by selenocysteine availability, termina tion occurs at the UGA codon. From these and similar data, it was clear that UGA in the message must correspond to selenocysteine in the selenopolypep tide even though amino acid sequence data were lacking. Recently, other glutathione peroxidase gene sequences have been determined, and all are highly conserved. The TGA codon was found in the expected position in human (70-72) and rat (73) glutathione peroxidase genes. Selenium-containing hydrogenases have been isolated from various sulfate reducing anaerobic bacteria, and the gene encoding the hydrogenase from D. bacu tatu s was cloned and sequenced (74, 32). A TGA codon detected near the end of the opcn reading frame of this gene indicates that the protein should contain selenocysteine, but this has not been isolated and identified. Howev er, based on the previously demonstrated occurrence of selenocysteine in a hydrogenase isolated from a methane-producing anaerobic organism, the assumption probably is correct.
E. coli and S. typhimurium Genes Affecting Selenium Metabolism A collection of E. coli and S. typhimurium mutants that were selected on the basis of inability to synthesize selenium-dependent formate dehydrogenases and seleno-tRNAs have proven invaluable in elucidating essential steps in the selenium incorporation processes. Whereas some of the mutations prevented the incorporation of selenium into the selenopolypeptides of the formate dehydrogenases but had no effect on selenation of tRNAs, a lesion in one gene resulted in the loss of ability to insert selenium in both types of macromolecules. These mutants, originally assigned to genetic classes desig natedfdhA, fdhB, andfdhC (75), or selAl (60), have been reclassified and the genes are now referred to as setA, setB, setc, and seLD (69). The setA, setB, and seLD gene products are proteins and the selC gene product is a new tRNA (69, 76-78). Although no mutants have been reported that lack the
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ability to convert 2-thiouridine residues of tRNAs to 2-selenouridines but still retain the ability to insert selenium into formate dehydrogenase selenopoly peptides, an asuE mutant that lacks the 2-thiouridines (79) also lacks 2selenouridines in tRNAs (60). Since this strain maintained ability to in corporate selenium into formate dehydrogenase, it was concluded that seleno tRNA does not serve as a source of selenium for selenoprotein synthesis (60). Furthermore, the precursor role of 2-thiouridine residues in tRNAs for seleno tRNA synthesis is supported. tRNAs With Anticodons Complementary to UGA A new tRNA discovered in E. coli that is the product of the selC gene (76) and a mammalian UGA suppressor tRNA formerly designated as phosphoseryl tRNA (80, 81) have anticodons that recognize the UGA codon and are aminoacylated with L-serine by seryl-tRNA ligase. The esterified L-serine residues are phosphorylated by specific kinases (82, 83), and the resulting o-phosphoseryl-tRNAs are then converted to selenocystyl-tRNAs (84, 85) (Scheme 2). Thus, in these cases a performed selenocysteine is not esterified directly, but rather it is generated in situ from a serine residue initially esterified to the tRNA. In E. coli two gene products that are required for this process have been identified (69, 77, 84). One of these, the selA gene product, is a 50,OOO-dalton protein that may be the kinase, and the other is the seLD gene product, a 37,OOO-dalton protein presumably functioning in the selenium substitution step. The in vivo formation of selenocystyl-tRNA has been demonstrated both in E. coli (84) and in cultured rat mammary tumor cells (85). The requirement of a functional selC gene for synthesis of the
:
Generation of Se-Cys-t-RNA
�
+�.. "
' '"'
OH P O·C HC-CH20H NH,
e
1::..
l'''':::l
I '";"' ._& � ''H' I .& 'H p P OH .
'R-Se
qH He-CHo! Q-p=O NH, a-
Enzyme
.•�. 'o.:c
OH
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..."'"'
O. C HC-CH 2 SeNH,
Scheme
2
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selenopolypeptides of formate dehydrogenase in E. coli provides evidence that the role of the new tRNA is to deliver selenocysteine to the UGA site on the message for incorporation into the growing polypeptide chain. Presum ably the corresponding mammalian tRNA has the same function. The anticodons of the E. coli (76, 77) and the eukaryotic tRNAs (77, 86, 87), as deduced from the gene sequences, are UCA, but in some mammalian tRNAs the uridine in the "wobble position" of the anticodons is modified posttranscriptionally to a 2' -o-methylcytidine or to an unidentified uridine derivative (80). The 2' -o-methylcytidine containing tRNA appears to be the species that is active in selenocystyl-tRNA formation (D. Hatfield, personal communication).
The selB Gene Product is a New GTP-Binding Translation Factor The seLB gene product, a 68,000-dalton protein, has been shown to bind guanine nucleotides and to interact specifically with selenocystyl-tRNASer but not with seryl-tRNASer (78). Thus, the probable role of this protein is to serve as an elongation factor specific for selenocysteine. As one measure of com plex formation between the SELB protein and selenocystyl-tRNASer, the ability to protect against deesterification by alkaline hydrolysis or against tRNA cleavage by ribonuclease was tested. In both assays the SELB protein contributed significantly to the stability of the aminoacylated derivative and to the tRNA itself when the tRNA was in the form of selenocystyl-tRNAs er, but no protection was afforded when it was tested as seryl-tRNAs er. The addition of guanine nucleotides was not required for the effects observed. A compari son of the binding constants exhibited by the SELB protein and EF-Tu for guanine nucleotides indicated similar values for GTP but a much lower affinity for GDP by the SELB protein. Amino acid sequence homology of the amino-terminal portion of the SELB protein with the G-protein family in dicated the portion of the molecule involved in guanine nucleotide binding.
Role of the selD Gene Product Mutants of S. typhimurium (60) and E. coli (69) unable to incorporate selenium specifically into formate dehydrogenase and tRNAs were shown to be defective in a single gene that is now termed selD (69,88). This gene maps at about 21 minutes on the S. typhimurium chromosome and at about 38 minutes on the E. coli chromosome. The product of this gene is a 37,000dalton protein that is required for the conversion of seryl-tRNASer to selenocystyl-tRNASer (77, 84). Although this fact explains the requirement of the selD gene product for biosynthesis of the formate dehydrogenase seleno polypeptide, it does not account for the role of this gene product in the synthesis of a 2-selenouridine residue in tRNA. Because the generation of a
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reactive sdcnium donor could be the common requirement for the two processes, it is possible that the SELD protein fulfills such a role. Replace ment of the activated serine hydroxyl group and an activated sulfur in 2-thiouridine by selenium could thus be controlled by a single gene product. As discussed later, the conversion of an esterified serine in subtilisin to selenocysteine by reaction with a high concentration of hydrogen selenide at elevated temperature (89), together with the known reactivity of the 2thiocyanate derivative of uridine with hydrogen selenide to form 2selenouridine ( 90), may be models for the enzymatic processes. An entirely unexpected effect of a mutation in the seLD gene was observed in a S. typhimurium mutant, selAJ (60). This organism incorporated selenocysteine into numerous proteins of the cell even though it was unable to synthesize the specific selenopolypeptides of formate dehydrogenase (60, 88). In addition to efficient uptake of selenium and incorporation in proteins as selenocysteine, which accounted for 20% of the selenium added to the culture medium, some selenomethionine was incorporated. Selenium containing proteins of an E. coli seLD mutant accounted for 30% of the selenite (0.5 I-tM) provided in the culture medium. In this organism sele nomethionine was the major selenoamino acid in the proteins, and a lesser amount of selenocysteine was present. From these results it is clear that a mutation in the seLD gene does not affect an early step in the pathway of selenium uptake and metabolism. Furthermore, a pathway of synthesis of the selenoamino acids that is specific for selenium and unrelated to the synthesis of selenocystyl-tRNAser is present in these bacteria. Whether the nonspecific incorporation of selenocysteine in these bacterial proteins, in what appears to be an entirely random fashion, is more widespread than currently believed or is a consequence of the seLD mutation is not presently known.
RELATIVE EFFECTIVENESS OF SELENIUM VERSUS SULFUR IN FORMATE DEHYDROGENASE When the complete E. coli formate dehydrogenasejdhF gene in which TGA had been mutagenized to TGC or TGT was expressed in an E. coli host lacking the selA and selB genes (fdhA) , formate dehydrogenase activity was still detectable in the cells. In contrast, conversion of TGA to TeA ( serine) completely abolished enzyme activity. Replacement of selenocysteine with cysteine in formate dehydrogenase by site-specific mutagenesis was estimated to result in a 75-80% decrease in activity of the enzyme ( M. J. Axley, T. C. Stadtman, and A. Bock, unpublished). These otherwise identical enzymes thus provide biological material for more exact assessment of the advantage of a selenol group that is largely ionized at physiological pH over a sulfhydryl group with its higher pKa in a redox catalyst. However, since both the sulfur
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and the selenium enzymes are highly oxygen labile, preparation of fully active species is difficult. A comparison (77) of the sequences of the fdhF gene of E. coli that encodes a selenocysteine-containing protein and the significantly homologous formate dehydrogenase gene from M. formicicum (19) indicates that substitution of TGC for TGA has occurred in nature. The high concentra tion of the non-selenium-containing enzyme in M. formicicum apparently compensates for the lower activity of a cysteine redox center as compared to a selenocysteine redox center.
CHEMICAL SYNTHESIS OF SELENOCYSTEINE-CONTAINING ENZYMES Two approaches have been used for the construction of selenocysteine containing enzyme catalysts. In one case a 25-amino-acid polypeptide was synthesized by the Merrifield method and selenocysteine residues were in serted directly (91). The protein prepared in this way is the copper metal lothionein of Neurospora crassa which, in the native form, contains seven cysteine residues (92). For the selenoprotein a selenocysteine residue was introduced at each of the seven positions normally occupied by cysteine. The protein containing only cysteine was prepared in the same way. A brief report of metal binding and physical chemical properties of the two types of metal lothioneins indicated many similarities (91). Detailed studies on the relative effectiveness of sulfur versus selenium in a protein designed to transport or detoxify metals should provide interesting data. A different method was used to introduce selenocysteine into the active site of subtilisin, thus converting the protease into an acyltransferase. By adapting a method previously used for conversion of the active site serine of subtilisin to cysteine (93), a new catalyst termed selenolsubtilisin that contained sele nocysteine in place of the serine was produced (89). In this procedure the serine hydroxyl group was selectively activated by reaction with phenyl methanesulfonyl fluoride, and the sulfonylated enzyme was treated with 0.5 M hydrogen selenide in aqueous pH 6.8 buffer for 36 hours at 40°C. Yields of 40-50% were reported for selenoenzyme formation. Studies of the properties of selenolsubtilisin indicated it to be a poor catalyst of amide hydrolysis, whereas reactivity with esters and subsequent transfer of the covalently bound acyl group to water or to amines was greatly accelerated.
ENZYMES THAT DEGRADE SELENOCYSTEINE AND SELENOCYSTINE Two pyridoxal phosphate-dependent enzymes, one termed L-selenocysteine p-Iyase and the other D-selenocystine a,p-Iyase, have been isolated in
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homogeneous fonn and characterized. The enzyme active on L-selenocysteine is widely distributed in mammalian tissues and in bacteria and was isolated from pig liver (94) and from Citrobacter freundii (95). The selenoamino acid is decomposed in the presence of dithiolthreitol to give stoichiometric amounts of hydrogen selenide and L-alanine. When the reaction was carried out anaerobically in the absence of dithiolthreitol, elemental selenium was detected as the product of the J3-elimination reaction. The enzyme from pig liver, Mr 85,000-90,000, is a dimer of 48,000 Mr subunits; the C. freundii enzyme is a monomeric protein of 64,000 Mf' The enzymes also differ in amino acid composition and several physicochemical properties. However, they are similar in their enzymological properties, and both are inactivated by J3-chloro-L-alanine, a mechanism-based inactivator. Although it is suggested that the physiological role of L-selenocysteine-J3-lyase could be detoxification of the selenoamino acid to elemental selenium, there is no clear-cut evidence on this point. The high Km value (about 1 mM) exhibited by the enzyme from both mammalian and bacterial sources for selenocysteine could be consistent with a detoxification role. The pYlidoxal phosphate-dependent o-selenocystine-a,J3-lyase was de tected in certain anaerobic amino acid fennenting bacteria and was isolated from C. sticklandii (96). This enzyme, Mr 74,000, consists of two identical 35,000 Mr subunits. D-cystine and D-cysteine also serve as substrates, but L-amino acids are inert. The enzyme, although resembling D-cysteine de sulfhydrase of E. coli, was distinguished from the latter by showing that there was no cross-reactivity with antibodies elicited to the two pure enzymes. The products fonned from D-selenocystine by this novel enzyme are pyruvate, ammonia, and elemental selenium. The initial elimination product is thought to be the perselenide of D-selenocysteine (R-Se-Se-). Although a reactive perselenide is attractive as a potential selenium donor for selenoprotein and seleno-tRNA biosynthesis, the enzyme has not been detected as a participant in the processes nor has any clue been found as to its biological role.
DETOXIFICATION OF SELENIUM COMPOUNDS BY METHYLATION The role of methylation as a means of detoxification of selenium compounds is well known as dimethylselenide and trimethylselenonium ion are common excretory products (9, 10). Generation of dimethylselenide, following reduc tion of ingested selenite to hydrogen selenide, converts the highly toxic selenium compounds to a volatile, less toxic fonn. This series of reactions also is catalyzed by a number of fungi. Although it has been generally accepted that the methylated derivatives are dead end products, there is newer evidence to show that demethylation processes are important and recycling of selenium from these compounds occurs (97).
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Literature Cited I. Stadtman, T. C. 1974. Science 183:9152. 3. 4.
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5.
6. 7.
8.
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