Selenoproteins in Archaea and Gram-positive bacteria

Biochimica et Biophysica Acta 1790 (2009) 1520–1532

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Review

Selenoproteins in Archaea and Gram-positive bacteria Tilmann Stock, Michael Rother ⁎ Molekulare Mikrobiologie und Bioenergetik, Institut für Molekulare Biowissenschaften, Goethe-Universität Frankfurt am Main, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany

a r t i c l e

i n f o

Article history: Received 28 February 2009 Received in revised form 23 March 2009 Accepted 23 March 2009 Available online 31 March 2009 Keywords: Selenium Selenocysteine Selenoprotein Clostridium Moorella Eubacterium Methanococcus maripaludis

a b s t r a c t Selenium is an essential trace element for many organisms by serving important catalytic roles in the form of the 21st co-translationally inserted amino acid selenocysteine. It is mostly found in redox-active proteins in members of all three domains of life and analysis of the ever-increasing number of genome sequences has facilitated identification of the encoded selenoproteins. Available data from biochemical, sequence, and structure analyses indicate that Gram-positive bacteria synthesize and incorporate selenocysteine via the same pathway as enterobacteria. However, recent in vivo studies indicate that selenocysteine-decoding is much less stringent in Gram-positive bacteria than in Escherichia coli. For years, knowledge about the pathway of selenocysteine synthesis in Archaea and Eukarya was only fragmentary, but genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has not only led to the characterization of the pathways but has also shown that they are principally identical. This review summarizes current knowledge about the metabolic pathways of Archaea and Gram-positive bacteria where selenium is involved, about the known selenoproteins, and about the respective pathways employed in selenoprotein synthesis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction For the longest time since the discovery of selenium this element was regarded as highly toxic [1] and only in 1954 was the importance of selenium for the synthesis of enzymes involved in formate oxidation of Escherichia coli recognized [2]. In the following decades until today much scientific effort has been spent to understand the biology of selenium and we know now that it is a trace element essential for many organisms including humans [3–5]. It was found to be a constituent of a base modification (5-[(methylamino)methyl]-2selenouridine) in certain tRNAs [6,7] derived by specific substitution of selenium for sulfur in 2-thiouridine, which is catalyzed by 2selenouridine synthase (the ybbB gene product in E. coli) [8]. Much less is known about non-covalently bound selenium-containing cofactors found in anaerobic xanthine and nicotinate dehydrogenases. The most important – and best-characterized – biological form of selenium is that of the amino acid selenocysteine (Sec, 2-selenoalanine). As the name suggests, it is structurally identical to cysteine (Cys), only with the thiol group replaced by a selenol group. Sec was discovered as a unique amino acid in 1976 [9], but only in 1986 it was found that Sec is co-translationally inserted into growing polypeptides at the position of an in-frame UGA (opal) nonsense codon on the mRNA [10,11]. It was therefore designated as the 21st genetically encoded amino acid [12]. Why organisms contain this unusual amino acid is still not completely understood but probably its physicochemical properties are exploited [13]. The use of Sec might, thus, ⁎ Corresponding author. Tel.: +49 69 79829320; fax: +49 69 79829306. E-mail address: [email protected] (M. Rother). 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.03.022

partly be explained by its high nucleophilicity and the fact that the selenol group is mostly deprotonated at physiological pH due to its lower pKa value (5.2 for Sec, 8.3 for Cys) making it more reactive than Cys [14,15]. Therefore, it is almost exclusively found in the catalytic site of numerous redox-active enzymes. However, for most characterized selenoproteins the specific functions of Sec are still unknown because for all but one (see below) of the selenoproteins homologous proteins with Cys at the respective position exist in other organisms [16]. Consequently, not all organisms require selenium. In fact, the majority of known organisms get by well without employing Sec and the evolution of the Sec-encoding trait has been the subject of much debate (see for example [17] and [18] and references therein). Since this trait is present in members of all three domains of life with some fundamental commonalities conserved, it now appears that Sec was a late addition to the genetic code but emerged before the division of the three domains [19], i.e. it must have been present in the last universal common ancestor. Subsequently, the Sec-trait was possibly lost in many lineages during evolution and some lineages later regained it through lateral gene transfer [20]. The pathway of Sec biosynthesis and incorporation of E. coli was the first to be unraveled and many of the discoveries there were made in the laboratory of August Böck. Since this pathway is comprehensively reviewed elsewhere (Yoshizawa and Böck, this issue, [21–23]) it will only be summarized briefly here (Fig. 1). First, a Sec-specific tRNA (tRNAsec, the selC gene product) is charged with serine by seryl-tRNA synthetase (SerRS), and the seryl moiety is subsequently converted to a selenocysteyl-moiety by Sec synthase (the selA gene product). The selenium donor for this reaction is selenomonophosphate generated from a reduced selenium species by selenophosphate synthetase (SPS,

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Fig. 1. Sec biosynthesis and incorporation in E. coli. [Se], reduced Se-species; SelB, Sec-specific elongation factor; SerRS, seryl-tRNA synthetase; SPS, selenophosphate synthetase; SS, Sec synthase; see text for details; scheme adapted from [23].

the selD gene product). The specialized translation elongation factor SelB (the selB gene product) delivers the selenocysteylated tRNA to the A site of the ribosome. SelB is homologous to the canonical elongation factor EF-Tu in its N-terminal part but carries a C-terminal extension responsible for binding the SECIS (selenocysteine insertion sequence) element. The SECIS element, a secondary structure on bacterial selenoprotein mRNAs, located directly downstream of the UGA codon, triggers a conformational change in the GTP•SelB•Sec-tRNAsec complex which allows for insertion of the charged tRNA into the ribosomal A site. In this review we aim to summarize the current knowledge about selenoproteins in Gram-positive bacteria and in Archaea and the respective pathways of Sec synthesis and incorporation into proteins. Although these two groups of microorganisms are phylogenetically unrelated, many of their respective selenoproteins are active in conceptually similar metabolic pathways. The two microbial groups also have in common that until recently, considerable gaps existed in the knowledge about the respective factors required for synthesis and incorporation of Sec. However, this has changed dramatically, especially for the Archaea. Identification and characterization of the reactions leading from tRNA-bound serine to Sec in Methanococcus and mammals showed that Archaea and Eukarya employ identical pathways. This underscores that Archaea are not just “strange

bacteria” but – as the phylogenetic tree of life illustrates [24] – in many of their features can be regarded as “simple eukaryotes”. 2. Pathways involving selenoproteins in Gram-positive bacteria The Gram-positive bacteria were originally classified by their staining characteristics based on the organism's thick peptidoglycan layer and the lack of an outer membrane found in Gram-negative bacteria [25]. As a result of accumulation of new strains and the advent of molecular phylogenetic techniques the Gram-positives are now sub-divided into Actinobacteria (high G + C Gram-positives including, e.g. actinomycetes and mycobacteria), Tenericutes (cell wall-less bacteria including, e.g. mycoplasmas), and Firmicutes (low G + C Gram-positives including, e.g. the bacilli and clostridia). All Gram-positive bacteria for which selenoproteins have been experimentally demonstrated belong to the clostridial clade and are therefore strictly anaerobic. Table 1 lists prominent examples, their life style, and the number of (putative) selenoproteins they encode. Members of this clade are able to grow by means of various mixed acid and alcohol fermentations (e.g. butyrate-, acetone/butanol-, Stickland-fermentation) and/or anaerobic respirations (e.g. sulfate- or acetogenic carbonate respiration) and are either obligate or facultative heterotrophs [26,27]. Metabolic pathways where selenoproteins are

Table 1 Gram-positive bacteria with selenium-dependent proteins. Organism Moorella thermoacetica Eubacterium acidaminophilum Clostridium sticklandii Clostridium difficile Clostridium acidiurici Clostridium barkeri Clostridium purinolyticum Clostridium kluyveri Carboxydothermus hydrogenoformans a b c

Life style Acetogenesis via acetyl-CoA pathway Glycine/Stickland fermentation Stickland fermentation Stickland fermentation Purine fermentation Purine/pyrimidine fermentation Glycine/purine fermentation Ethanol-acetate fermentation CO-dependent hydrogenogenesis

Number of selenoproteinsa Exp.b

Pred.c

1 7 6 4 2 (1) (1) (1) 0

2 6 0 1 0 0 0 0 12

Non-Sec-containing, selenium-dependent proteins are indicated in brackets. Verification either by radioactive labeling, determination of selenium in the protein, or correlation of gene sequence and protein. Selenoproteins predicted from DNA sequence data in addition to known ones (where applicable).

Reference [96,57] [91,72] [74] [175,176] [177] [52] [178] [179,180] [181]

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involved include autotrophic acetogenesis via the reductive acetylcoenzyme A (acetyl-CoA) pathway, glycine-dependent acetogenesis, acetogenesis via Stickland-fermentation, and purine/pyrimidinedependent acetogenesis (Figs. 2–4). Bacteria employing the reductive acetyl-CoA (also known as Wood–Ljungdahl) pathway for energy conservation are a phylogenetically diverse group of obligate anaerobes forming acetate from CO2 or from organic substrates (for reviews see [28–30]). The most important model organisms in elucidating this pathway and its bioenergetics were/are Moorella thermoacetica (formerly Clostridium thermoaceticum) and Acetobacterium woodii [31]. Although discovered and most extensively studied in Gram-positive acetogens, Gramnegative Spirochetes such as Treponema primitia, are apparently involved in H2-dependent, and thus autotrophic, acetogenesis via the Wood–Ljungdahl pathway in the hindguts of termites [32,33]. The acetyl-CoA pathway is also present in a (still increasing) number of non-acetogens, mainly as a means for autotrophic carbon assimilation [34,35]. Because it serves both energy conservation and carbon assimilation, because the C1-transfer reactions involve metals thought to be involved in the autotrophic origin of life [36], and because it is conceptually very similar to the pathway of methanogenesis (see below), the Wood–Ljungdahl pathway is believed to be one of the first types of metabolism on earth [37]. In the “methyl-branch” of the Wood–Ljungdahl pathway (Fig. 2), CO2 is first reduced to formate by formate dehydrogenase (FDH) which uses reducing equivalents derived from the oxidation of molecular hydrogen catalyzed by hydrogenase. The former enzyme is often a selenoprotein (see below). Formate is then activated to N10formyl-tetrahydrofolate (formyl-H4F) at the expense of ATP hydrolysis by formyl-H4F synthetase. The formyl-group is subsequently converted to a methyl-group by dehydration to N5,N10-methenyl-H4F (by formyl-H4F cyclohydrolase) and reduction of N5,N10-methenyl-H4F via N5-methylene-H4F (by methylene-H4F dehydrogenase) to N5-methylH4F (by methylene-H4F reductase). The methyl-group is then transferred via a corrinoid- and Fe/S cluster-containing protein to the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) to form acetyl-CoA with CoA and enzymebound CO. In the “carbonyl-branch” of the pathway (Fig. 2), CO2 is reduced to COCO2 by CODH to provide the carbonyl-group of acetylCoA. Acetyl-CoA is then converted to acetate via acetyl-phosphate by phosphotransacetylase and acetate kinase [38,39], which yields ATP. Thus, formation of acetate from H2 + CO2 results in no net ATP synthesis via substrate-level phosphorylation (SLP) (Fig. 2) but it has been shown that this path is coupled to the formation of an ion motive force across the cytoplasmic membrane allowing for synthesis of ATP via a chemiosmotic mechanism [31]. However, which steps in acetogenesis from CO are coupled to ion extrusion is still not clear yet, but efforts are currently made to close this gap in knowledge [40]. Another energy-yielding pathway of peptidolytic and purinolytic clostridia involving selenoproteins is the fermentation of amino acids such as glycine, methylated glycine derivatives (sarcosine and betaine), and purines (Figs. 3, 4). Examples of organisms capable of growing on glycine as the sole energy source are Clostridium purinolyticum and Eubacterium acidaminophilum [41]. There, glycine is reductively deaminated to acetyl-phosphate by Sec-containing glycine reductase. Acetate formation from acetyl-phosphate via acetate kinase yields ATP for growth. Oxidized glycine reductase is re-reduced by the thioredoxin/thioredoxin reductase system [42] with the reducing equivalents derived from glycine oxidation (Fig. 3). Glycine decarboxylase/synthase splits glycine in a pyridoxal-phosphate (PLP)-dependent manner to an enzyme-bound S-aminomethyl group, which is then oxidatively deaminated and transferred to H4F yielding reduced NADP+ and methylene-H4F. The latter provides the cell with C1 compounds for anabolic purposes or is further oxidized to CO2 via the “methyl-branch” of acetogenesis running in reverse, which yields two reducing equivalents (Fig. 3). However, some organisms

Fig. 2. The reductive acetyl-CoA (Wood–Ljungdahl) pathway of acetogenesis. Acetyl-P, acetyl-phosphate; [CO], enzyme-bound CO; [Co-Fe/S], corrinoid/iron–sulfur protein; FDH, formate dehydrogenase; H2ase, hydrogenase; H4F, tetrahydrofolate; HS-CoA, coenzyme A; X, arbitrary electron acceptor; 2, formyl-H4F synthetase; 3, formyl-H4F cyclohydrolase; 4, methylene-H4F dehydrogenase; 5, methylene-H4F reductase; 6, methyltransferase; 7, carbon monoxide dehydrogenase; 8, acetyl-CoA synthase; 9, phosphotransacetylase; 10, acetate kinase; selenoproteins involved are round-boxed in orange, the Cys-containing isoforms are square-boxed in white; the potential involvement of a Sec-containing hydrogenase is indicated by the dashed round box; see text for details; scheme adapted from [29].

such as C. sticklandii or C. sporogenes can only grow on pairs of amino acids via a Stickland-type fermentation [43], with one (e.g. alanine) being oxidized to the corresponding acyl-CoA derivative (acetyl-CoA) while the other (e.g. glycine) is being reduced (e.g. to acetate as outlined above). The former process is coupled to ATP synthesis via SLP and generates reduced pyridine nucleotides, which are reoxidized in the latter; ATP synthesis via SLP only occurs in the case of glycine (see above). For example, during fermentation of alanine and proline, reduction of D-proline to 5-aminovalerate is catalyzed by a membrane-bound, Sec-containing D-proline reductase in C. sticklandii [44], which is not further coupled via SLP to ATP synthesis. Instead, this fermentation appears to be coupled to proton transfer across the cytoplasmic membrane via an unknown respiratory chain and, thus, may involve ATP synthesis by a chemiosmotic mechanism [45]. Betaine (N,N,N-trimethylglycine) and sarcosine (N-methylglycine) can be metabolized by different routes. In one path, betaine reductase or sarcosine reductase (both selenoproteins) reductively deaminate betaine or sarcosine (in completely analogous reactions to glycine reductase) to acetyl-phosphate (see above) and trimethylamine or monomethylamine, respectively (Fig. 3). Acetyl-phosphate yields ATP by SLP (see above) and the methyl-groups from the methylamines can be transferred via corrinoid-containing methyltransferases to H4F.

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leading to glycine and N5-forminino-H4F, which can subsequently be deaminated to N5,N10-methenyl-H4F, an intermediate in both the Wood–Ljungdahl and the glycine pathway (see above). Oxidation of N5,N10-methenyl-H4F to CO2 (via formate and Sec-containing FDH) provides reducing equivalents for reduction of N5,N10-methenyl-H4F to glycine, and eventually acetate (Fig. 4). If reducing equivalents are generated by the oxidation of hypoxanthine to xanthine, formate is not oxidized but excreted [50]. A number of bacteria also ferment pyrimidines such as uracil, orotic and nicotinic acid. Degradation of the latter is initiated by nicotinate dehydrogenase, which is similar to xanthine dehydrogenase and also contains non-covalently bound selenium [51]. The complete catabolic pathway in E. barkeri from nicotinate to pyruvate and propionate has recently been elucidated which showed that the glycine pathway of acetogenis is not involved [52]. 3. Selenoproteins in Gram-positive bacteria As shown in the previous section selenoproteins are involved in numerous energy-yielding pathways of Gram-positive bacteria. However, selenoproteins are also found in these organisms to be involved in other processes, such as antioxidant defense, redox cycling and redox homeostasis, and even Sec biosynthesis itself. Furthermore, comparative genome analyses have identified a number of “new”

Fig. 3. The glycine-dependent path of acetogenesis. Acetyl-P, acetyl-phosphate; BR, betaine reductase; [CO], enzyme-bound CO; DMA, dimethylamine; GR, glycine reductase; H4F, tetrahydrofolate; MMA, monomethylamine; SR, sarcosine reductase; TMA, trimethylamine; 2–10, see Fig. 2; 11, glycine decarboxylase/synthase; the dashed arrows represent methyl-transfer reactions; selenoproteins involved are round-boxed in orange, the Cys-containing isoforms are square-boxed in white; scheme adapted from [41].

Methyl-H4F is then either oxidized to CO2 to provide reducing equivalents, or converted to acetate, either via acetyl-CoA and reactions involving CODH/ACS (see above) or via glycine involving glycine decarboxylase/synthase and glycine reductase (Fig. 3). Alternatively, the methyl-groups of betaine and sarcosine can (successively) be transferred to H4F yielding methyl-H4F and (eventually) glycine, which can both be further metabolized as described above. As can be seen from Fig. 3 the intermediates methylene-H4F, methyl-H4F, glycine, acetyl-CoA and acetyl-phosphate are inter-convertible, which makes it often difficult to predict what route(s) will be used by a given organism. Members of the purinolytic clostridia (e.g. C. acidiurici, C. cylindrosporum, C. barkeri, C. purinolyticum) readily ferment purines to CO2, ammonia, glycine (or further to acetate), and formate. Purine, adenine, guanine, uric acid and their derivatives are all converted (via hypoxanthine) to the common intermediate xanthine [41] involving, depending on the substrate, xanthine dehydrogenase and purine hydroxylase [46,47]. Both enzymes are molybdo-flavoenzymes of the molybdenum hydroxylase class widespread in nature [48]. Both enzymes contain non-covalently bound selenium, which can be removed by cyanide [49]. Since the molybdenum atom in molybdopterin is coordinated by a dithiol inserted during cofactor biogenesis [48] it is thought that selenium can be inserted instead of sulfur, which leads to greatly enhanced activity of the enzyme [49]. Xanthine is then converted to formiminoglycine by a series of deaminations and decarboxylations [50]. The formimino-group is transferred to H4F

Fig. 4. Selenium-dependent purine degradation of clostridia. [CO], enzyme-bound CO; [Co-Fe/S], corrinoid/iron–sulfur protein; FDH, formate dehydrogenase; HS-CoA, coenzyme A; PH, purine hydroxylase; XDH, xanthine dehydrogenase; 2–11, see legends to Figs. 2, 3; 12, glycine formimino transferase; 13, formimino-H4F cyclodeaminase; selenoproteins involved are round-boxed in orange, the Cys-containing isoforms are square-boxed in white; proteins containing non-Sec labile selenium are shown in dashed red; the dashed arrows depict series of reactions leading to xanthine and formiminoglycine, respectively; see text for details; scheme adapted from [50].

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Table 2 Selenium-containing proteins of Gram-positive bacteria. Protein Sec-containing Formate dehydrogenase Glycine reductase PA Glycine reductase PB Sarcosine reductase PB Betaine reductase PB Proline reductase PB SPS Peroxiredoxin Methionine sulfoxide reductase not Sec-containing Xanthine dehydrogenase Purine hydroxylase Nicotinic acid dehydrogenase

Characteristic organism

Verified?a

Reference

M. thermoacetica C. sticklandii E. acidaminophilum E. acidaminophilum E. acidaminophilum Clostridium sticklandii E. acidaminophilum E. acidaminophilum Clostridium OhILAs

Yes Yes Yes Yes Yes Yes Yes Yes Yes

[57] [9] [67] [71] [182] [74] [92] [80] [78]

C. acidiurici C. purinolyticum C. barkeri

Yes Yes Yes

[49] [46] [52]

a Verification either by radioactive labeling, determination of selenium, or correlation of gene sequence and protein.

selenoproteins based on the presence of (i) factors for selenoprotein synthesis in the respective organism, (ii) a TGA codon in the reading frame of the potential selenoprotein gene, and (iii) homologous genes in the database in which Cys is encoded at the respective position [53]. However, for most of these potential selenoproteins it is not known if they are synthesized, let alone what function they serve in the respective organism. Table 2 lists known selenium-dependent proteins in Gram-positive bacteria and the properties of the selenoproteins are summarized in the following section. 3.1. Formate dehydrogenase Formate dehydrogenases are widely distributed among the Bacteria and Archaea and the enzymes differ considerably in composition, properties and types of electron acceptors utilized. FDH catalyzes the reversible oxidation of CO2 to formate and is involved in energy metabolism, carbon fixation and pH homeostasis [54]. It contains Fe/S cluster and either Mo or W [55] coordinated by a pterin cofactor. E. acidaminophilum contains only W-dependent FDH [56], but these elements seem somewhat interchangeable in anaerobic organisms, as the enzyme from M. thermoacetica is active in its Mo- and W-form; however, the latter leads to higher activity [57]. NADP+ is the natural electron acceptor for FDH in M. thermoacetica [58], while the enzyme from C. pasteurianum has been shown to utilize ferredoxin [59]. The natural electron donor for FDH of E. acidaminophilum or C. purinolyticum still remains to be identified. The first prokaryotic gene for which Sec was shown to be encoded by TGA was that of the membrane-bound, formate:hydrogen lyaselinked FDH (fdhF) from E. coli [11]. The structure of the protein revealed that Sec coordinates the Mo of the pterin cofactor and takes part in catalysis [60]. Importantly, FDH is neither restricted to anaerobic microbes nor is it always Sec-dependent [61,62]. Still, it is the most widely distributed selenoprotein found in nature and it has been suggested that the genes encoding FDH, together with the genes encoding the machinery for Sec synthesis and incorporation, were extensively transferred laterally [20]. 3.2. Glycine reductase As outlined above, the glycine reductase system is the key for acetate formation via glycine and it was long thought to be exclusively distributed among clostridia. However, genome sequence analyses as well as physiological and immunological studies showed that at least one member of the Spirochetes, the oral pathogen Treponema denticola, also employs the glycine reductase system during growth via Stickland fermentation [63,64]. The systems of C. sticklandii and E. acidaminophilum have been studied in detail on the biochemical and molecular level (for reviews see [41,65,66]). It consists of three proteins, PA, PB, and

PC. PB is the substrate-specific enzyme catalyzing activation of glycine; it consists of three subunits, the Sec-containing GrdB and two polypeptides which derive from post-translational cleavage of the proprotein GrdE [67,68]. Activation of glycine proceeds via a Schiff-base formed by a carbonyl-group, located in GrdB, with glycine. Presumably, nucleophilic attack of the selenol from Sec forms a Se-carboxymethyl selenoether bound to GrdB; although direct evidence for the involvement of Sec in glycine activation is still lacking, the fact that corresponding subunits of all characterized substrate-specific PB proteins of glycine, betaine, sarcosine and proline reductase (GrdB, GrdF, GrdH, and PrdB), respectively, contain Sec, strongly argues for its involvement in catalysis [66]. Also, heterologously produced PB variants in which Sec was replaced by Cys are completely inactive [66]. Interestingly, search for proteins containing redox-active Cys residues in databases led to identification of homologous sequences in which the Sec codon is replaced by Cys codons [16]; however, it is completely unknown what functions these “Cys-variants” might serve. PA (GrdA) is a small acidic, redox-active protein, which accepts the carboxymethyl group from GrdB. It was the first protein for which Sec was demonstrated to be a constituent and it is still the only selenoprotein for which no Cys-containing homolog is known [9,63]. Upon transfer of the carboxymethyl group from PA to PC, the selenium in PA becomes oxidized resulting in a mixed selenylsulfide with the thiol of a conserved vicinal Cys. Concomitantly, the C2 unit is reduced and a PC-bound acetyl thioester is formed; the exact mechanism of this reaction is still unknown but possible scenarios have been discussed [66]. Oxidized PA is re-reduced by the thioredoxin/thoredoxin reductase system in E. acidaminophilum [42]. In this organism, PC is composed of the two tightly associated GrdC and GrdD proteins [69]. The former contains several conserved Cys residues, which are all proposed to be directly or indirectly involved in the carboxymethyl transfer reaction. Its similarity to β-ketoacyl-carrier protein synthase suggests a secondary transfer of the acetyl group from GrdC to GrdD by a transacetylase-type mechanism [70]. GrdD contains two conserved Cys residues and biochemical analysis of mutant proteins point to one of them as the site of the thiol from which the acetyl thioester is phosphorolytically liberated as acetyl-phosphate [70]. Sarcosine reductase and betaine reductase share their PA and PC components with glycine reductase but contain different substratespecific, Sec-containing PB proteins[71]. Activation of sarcosine is thought to proceed by a very similar mechanism as activation of glycine, except that methylamine instead of ammonia is released (Fig. 3). Like GrdE, the proprotein GrdG is post-translationally processed [72]. Betaine has to be activated differently (not via Schiff-base) due to permethylation of the amino group; instead, strong ionic interactions between substrate-specific PB and betaine are thought to sufficiently polarize the C–N bond facilitating the nucleophilic attack by the selenol anion of PA [41]. Interestingly, the proprotein GrdI of betainespecific PB is not post-translationally processed [72]. 3.3. Proline reductase L-proline is racemized to the D-form before it can be reduced to 5aminovalerate by C. sticklandii and C. sporogenes [44]. The reduction seems to proceed by a different mechanism compared to glycine reduction, although in both cases a carbon–nitrogen bond is cleaved [73]. Overall, D-proline reductase appears to be similar to PB of glycine reductase. The enzyme is composed of three different subunits [74]. PrdB contains Sec at a similar motif as found in GrdB of PB of glycine reductase. The proprotein PrdA is processed similar to GrdE and GrdG (see above) into two proteins [68].

3.4. Antioxidant defense proteins Although strictly anaerobic, members of the clostridia often contain enzymes for antioxidant defense. For example, methionine

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sulfoxide reductase (Msr) reduces oxidized methionine residues in proteins, which arise by the unwanted action of reactive oxygen species [75] (and see V. N. Gladyshev, this issue). MsrA is specific for the S-form of methionine sulfoxide [76,77], whereas MsrB is specific for the R-form. Clostridium sp. OhILAs employs a Sec-containing MsrB [78]. Both MsrA and MsrB can either be selenoproteins or nonselenoproteins, depending on the organism [78,79]. A Sec-containing peroxiredoxin from E. acidaminophilum was experimentally identified based on metabolic labeling with [75Se]-selenite, analysis of the encoding gene, and quantification of thiol-dependent peroxidase activity [80]. Metabolic labeling in conjunction with genome analysis further revealed that the organism also produces another, small selenoprotein of unknown function which contains a motif reminiscent of thioredoxin [72]. Furthermore, the presence of conserved Cys/Sec motifs found in glutathione/glutathione peroxidase/reductase, thioredoxin/thoredoxin reductase and related systems in putative selenoproteins derived from comparative genome analyses indicates the presence of numerous other Sec-containing, thiol-dependent oxidoreductases [63,81]. However, prediction of a (selenoprotein) gene allows no conclusion whether it is expressed or about its function in vivo [82]. 3.5. Selenophosphate synthetase SPS is encoded by the selD gene in E. coli and was shown to provide selenomonophosphate for Sec synthesis [83–85]. Characterization of the enzyme showed that SPS forms selenomonophosphate in a rather slow reaction by transfer of the γ-phosphate group of ATP to a reduced selenium species liberating AMP and orthophosphate via a phosphorylated enzyme-intermediate [86,87]. SPS homologs containing Sec have been identified in eukaryotes [88], Archaea [89], proteobacteria [90], and Gram-positive bacteria [63,72,91]. The E. acidaminophilum enzyme was experimentally shown to contain Sec [92], while no archaeal SPS homolog could be visualized by metabolic labeling with [75Se]-selenite, which was explained by the presumed low level of synthesis [89]. Therefore, the in vivo function of archaeal SPS still awaits to be demonstrated. What role the Sec residue plays during catalysis in these enzymes is not clear. Also, how Sec synthesis can be initiated employing an enzyme which itself is a selenoprotein, is a classic “chicken or egg” question; Haemophilus influenzae apparently solves this problem by initially operating SPS that is either devoid of Sec, or by forming Sec-tRNAsec in a selenophosphate-independent fashion [90]; however, no Sec-containing SPS was thus far investigated in greater detail. In eukaryotes, the Sec-containing SPS homologs (SPS2) have been shown to be involved in Sec synthesis while the Sec-independent forms (SPS1) are not [93,94]. 4. Selenoprotein synthesis in Gram-positive bacteria The pathway of Sec synthesis and incorporation in Gram-positive bacteria has been studied to appreciable detail on the biochemical and molecular level in M. thermoacetica and E. acidaminophilum. Both M. thermoacetica and E. acidaminophilum encode tRNAsec, Sec synthase, SPS, and SelB [91,95,96]. Since a genetic system is not established for either of these two organisms, in vivo analysis of factors involved in selenoprotein synthesis of Gram-positive bacteria has been conducted only in the heterologous host E. coli. Both the M. thermoacetica selA and selC complement the respective lesions in E. coli [95,97]. Thus, Gram-positive bacteria appear to employ the same overall strategy for Sec synthesis as E. coli. Recoding of the UGA Sec-codon is mediated by a sequence- and structure-specific interaction between the SECIS element and SelB but putative SECIS elements in Gram-positive bacteria are not as well defined as in E. coli. In fact, even in the same organism the different putative SECIS elements appear to be rather unrelated to each other [91]. Since the phylogenetic distance of organisms is also reflected by

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the degree of deviation in sequence and structure of the SECIS element (and thus the SECIS-recognizing domain of the cognate SelB) only SelBs and SECIS elements from closely related organisms could previously mediate Sec insertion in E. coli [98–100]. Surprisingly, selB of E. acidaminophilum also complements a selB lesion in E. coli, but only when its genuine tRNAsec is present [91], which indicates that SelB-dependent UGA recoding in Gram-positive bacteria proceeds via the same mechanism as in E. coli. However, the SECIS/SelB interaction effecting UGA recoding appears to be rather promiscuous as a SECIS element of Clostridium sp. was functional in E. coli, although at low efficiency [78]. Furthermore, the tRNAsec/SelB pair of E. acidaminophilum efficiently directs Sec-insertion into various selenoproteins from E. acidaminophilum in E. coli and UGA readthrough in translational fusions of lacZ with selenoprotein mRNAs from Desulfomicrobium baculatum, Campylobacter jejuni, and T. denticola, respectively, was also observed in this system [92]. The generally higher number of selenoproteins present (13 in E. acidaminophilum vs. 3 in E. coli) [72,101] might be the reason for this apparent promiscuity of the SECIS/SelB interaction in Gram-positive bacteria; possibly, this can be exploited in the future as a tool to produce selenoproteins from other bacterial species in E. coli. There has also been considerable progress in the understanding of the UGA recoding process in bacteria on the atomic level. Although the E. coli factors were known much longer and investigated to a much greater detail, a fragment of M. thermoacetica SelB derived from spontaneous proteolytic cleavage was the basis for the first highresolution structure of a trans-acting factor involved in selenoprotein synthesis [102]. The SECIS-binding C-terminal domain of M. thermoacetica SelB, both free and bound to a M. thermoacetica SECIS, as well as free SECIS, could be structurally solved allowing to propose a detailed model for the interaction of SelB with the SECIS element and the ribosome [103–106]. 5. Methanogenesis involves selenoproteins in Archaea Archaea were only recognized as a distinct phylogenetic group some 30 years ago [24] and were initially renowned for being strictly anaerobic and/or inhabiting inhospitable environments like solfataric hot springs, soda lakes, and submarine volcanic vents. However, Archaea are ubiquitous and constitute a significant portion of the global biomass [107]. The diversity of Archaea is vast but they share unique features not found in the other two domains, Bacteria and Eukarya [108]. Methane is the most abundant hydrocarbon present in our atmosphere and the only organisms producing (significant amounts of) methane are methanogenic archaea, a monophyletic lineage of the Euryarchaeota. In anaerobic environments lacking abundant electron acceptors such as sulfate or nitrate, methane is the final breakdown product of organic matter. Thus, methanogenesis, the biological formation of methane, plays an essential role in the global carbon cycle [109]. All methanogenic archaea investigated to date strictly rely on methanogenesis for energy conservation and, thus, growth. Principally, methanogenic substrates are converted to methane with the concomitant generation of an ion motive force across the cytoplasmic membrane, which can then be employed for ATP synthesis or other energy requiring cellular processes. The number of substrates utilized for methanogenesis is quite limited reflecting the narrow ecological niche methanogens occupy: most methanogens are only able to grow with H2 + CO2 (or formate, Fig. 5), some can utilize methylated compounds, and some can grow with acetate. These different substrate classes are metabolized via distinct, but overlapping, pathways (for reviews, see [110–115]). The only Archaea for which selenoproteins have been demonstrated, either by experimentation or prediction from genome sequence data are methanogens obligatory dependent on the

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Fig. 5. The path of CO2 reduction to methane in Methanococcus species. FdH2, reduced ferredoxin; FDH, formate dehydrogenase; FMD, formyl-methanofuran dehydrogenase; Fru, F420-reducing hydrogenase; H2F420, reduced coenzyme F420; H4MPT, tetrahydromethanopterin, HDR, heterodisulfide reductase; HS-CoB, coenzyme B (N-7-mercaptoheptanoyl-O-phospho-L-threonine); HS-CoM, coenzyme M (2-mercaptoethanesulfonic acid); MF, methanofuran; Vhu, F420-non-reducing hydrogenase; 2, formyl-MF:H4MPT formyltransferase; 3, methenyl-H4MPT cyclohydrolase; 4, F420-dependent methyleneH4MPT dehydrogenase; 5, H2-dependent methylene-H4MPT dehydrogenase; 6, methylene-H4MPT reductase; 7, methyl-H4MPT:HS-CoM methyltransferase; 8, methyl-CoM reductase; selenoproteins involved are round-boxed in orange, the Cys-containing isoforms are square-boxed in white; the dashed grey arrow indicates potential “electron bifurcation”; see text for details; scheme adapted from [110].

hydrogenotrophic pathway of methanogenesis [63,116]. However, by far not all hydrogenotrophic methanogens employ Sec. In this pathway, CO2 is sequentially reduced to methane in seven steps via coenzyme-bound intermediates using H2 as the electron donor (Fig. 5). If formate is the substrate, it is first reduced to CO2 via (sometimes Sec-containing) formate dehydrogenase (FDH) [54]. CO2 is reduced to the formyl-level and attached to methanofuran (MF, a 2-aminomethylfuran derivative) by formyl-MF dehydrogenase (FMD). The electron donor for FMD is reduced ferredoxin. The endergonic H2-dependent reduction of ferredoxin is driven by the ion motive force via a membrane-bound energy converting hydrogenase [117]. However, an alternative mechanism of H2dependent reduction of ferredoxin involving “electron bifurcation” during heterodisulfide reduction (see below) has recently been proposed [110]. The next two steps in hydrogenotrophic methanogenesis are the transfer of the formyl-group from MF to tetrahydromethanopterin (H4MPT, a cofactor functionally analogous to tetrahydrofolate), catalyzed by formyl-MF:H4MPT formyltransferase, and the subsequent conversion of N5-formyl-H4MPT to N5,N10-methenyl-H4MPT catalyzed by N5,N10-methenyl-H4MPT cyclohydrolase. All hydrogenotrophic methanogens analyzed to date contain a coenzyme F420dependent (F420 is a 5-deaza-ribolflavin derivative functionally analogous to pyridine nucleotides) N5,N10-methylene-H4MPT dehydrogenase (MTD), which catalyzes the reduction of N5,N10-methenyl-H4MPT with reduced F420 (F420H2) to N5,N10-methylene-H4MPT (Fig. 5). F420 is reduced by the F420-dependent hydrogenase, an oligomeric NiFe-enzyme. The subsequent step in hydrogenotrophic methanogenesis, reduction of N5,N10-methylene-H4MPT to N5-

methyl-H4MPT, also depends on F420H2 as the electron donor and is catalyzed by N 5,N 10-methylene-H4MPT reductase. However, reduction of N5,N10-methenyl-H4MPT to N5,N10-methylene-H4MPT involving the F420-dependent hydrogenase and MTD is also catalyzed in one step by the H2-dependent N5,N10-methyleneH4MPT dehydrogenase (HMD). HMD and MTD are up-regulated when nickel (or selenium for Methanococcus [118]) is limiting [119] and it has been proposed that under these conditions MTD actually operates in the opposite direction, generating F420H2 needed for the reductase-catalyzed reaction and for anabolic purposes, because the activity of F420-dependent hydrogenase, a Ni/Fe enzyme is compromised [120]. The next step in hydrogenotrophic methanogenesis is the transfer of the methyl-group from H4MPT to coenzyme M (HS-CoM, 2mercaptoethanesulfonic acid) catalyzed by the membrane-integral N5-methyl-H4MPT:CoM methyltransferase, which couples the exergonic methyl-transfer to sodium ion extrusion; thus, it functions as a primary sodium ion pump, which explains why all methanogens investigated to date strictly depend on the presence of sodium ions for methanogenesis [121,122]. The ultimate step in all methanogenic pathways is the reduction of methyl-CoM to methane catalyzed by methyl-CoM reductase. Methane and heterodisulfide (CoM-S-S-CoB) are generated from methyl-CoM and coenzyme B (N-7-mercaptoheptanoyl-O-phospho-Lthreonine, HS-CoB), which is the electron donor for this reaction (Fig. 5). CoM-S-S-CoB represents the terminal electron acceptor of an energy-conserving electron transport chain and reduction of CoM-SS-CoB with H2 as the electron donor involves (at least) two (sometimes Sec-containing) enzymes, F420-nonreducing hydrogenase and heterodisulfide reductase (HDR) [123,124]. Apparently, differences exist between the mechanisms employed by obligate (e.g. Methanococcus) and facultative (e.g. Methanosarcina) hydrogenotrophs, which have been reviewed elsewhere [110,115]. 6. Selenoproteins in Archaea It is striking that within the Archaea (according to at least 56 available genome sequences representing 43 genera), selenoproteins appear to be restricted to two genera, Methanococcus and Methanopyrus [63,116]. It is also intriguing that the verified selenoenzymes of methanogens, with the exception of selenophosphate synthetase, are all involved in hydrogenotrophic methanogenesis (Fig. 5). Genome sequence analyses, radioactive in vivo labeling, and mutational studies identified at least six methanogenesis-related selenoproteins in M. jannaschii, M. voltae, M. maripaludis, and M. kandleri [89,125–128]. Table 3 lists known and putative archaeal selenoproteins and their properties (where known) are summarized in the following section.

Table 3 Selenoproteins of Archaea. Selenoprotein

Subunit

Characteristic organism

Verified?a Reference

Formate dehydrogenase Formyl-methanofuran dehydrogenase F420-reducing hydrogenase F420-non-reducing hydrogenase Heterodisulfide reductase Selenophosphate synthetase HesB-like protein

FdhA

Methanococcus vannielii

Yes

[130,183]

FwuB

Methanopyrus kandleri

Yes

[127]

FruA

Methanococcus voltae

Yes

[138]

VhuD VhuU HdrA

Methanococcus voltae

Yes

[138,184]

a

Methanococcus jannaschii Yes

[89]

homomeric Methanococcus jannaschii No

[89]

unknown

[63]

Methanococcus jannaschii No

Verification either by radioactive labeling, determination of selenium in the protein, or correlation of gene sequence and protein.

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6.1. Formate dehydrogenase Archaeal FDH (Fig. 5) appears to be similar to the bacterial enzyme regarding its metal and pterin cofactor content (see above); however, the physiological role of FDH in methanogenic archaea is formate oxidation to CO2 rather than CO2 reduction and its physiological electron acceptor is F420, the reductant for two reactions during methanogenesis from CO2 [129]. FDH isolated from M. vannielii consists of a 66 kDa Sec-containing and a 33 kDa subunit and it was later shown that the organism contains a Sec-independent isoform as well [130]. 6.2. Hydrogenase Hydrogenases are widely distributed among bacteria, archaea and lower eukaryotes as the consumption of H2 provides organisms with a supply of reductant, which also may be used for energy conservation. Alternatively, H2 production enables organisms to dispose excess reductant in the absence of electron acceptors other than protons. Consequently, many microorganisms evolve H2 under strictly anaerobic conditions. The associated electron carriers vary greatly depending on the source of the enzyme, ranging from cytochromes to ferredoxins, nicotinamides, or other cofactors (see below). Today, three phylogenetically distinct classes of hydrogenases are known [131], the Ni-containing (Ni/Fe) hydrogenases [132], the iron-only (or Fe/Fe) hydrogenases [133], and the Fe/S cluster-free (formerly metalfree) hydrogenases [134]. The latter has so far been found exclusively in methanogenic archaea and it is present in most, except those belonging to the orders Methanomicrobiales and Methanosarcinales [112]. The vast majority of known hydrogenases fall into the former class which also harbors hydrogenases containing Sec in the active site. The crystal structures of Ni/Fe hydrogenase from sulfate reducing proteobacteria revealed the molecular geometry of Ni and Fe at the active site. Four Cys residues, or three Cys plus a Sec as shown for the enzyme from Desulfomicrobium baculatum [135], coordinate the Ni, with two of these Cys also binding the Fe atom. In addition, the Fe possesses three non-protein diatomic ligands, CN, CO, or SO, depending on the source of the enzyme. Although no Sec-containing hydrogenase from a Gram-positive bacterium has been described to date analysis of available genome sequences suggests that some (e.g. M. thermoacetica, C. hydrogenoformans) contain Sec-dependent hydrogenase(s) [63,96]. However, this still needs to be verified experimentally. On the other hand, Sec-dependent hydrogenases are well documented for methanogenic archaea. In fact, the first hydrogenase shown to contain Sec was from M. vannielii [136]. Hydrogenotrophic methanogens contain at least two types of hydrogenase, the coenzyme F420-reducing hydrogenase (Frh in Methanothermobacter, Fru and Frc in Methanococcus), and the coenzyme F420-non-reducing hydrogenase (Mvh in Methanothermobacter, Vhu and Vhc in Methanococcus). As the names suggest, the physiological electron acceptor for H2 oxidation of the former is F420 [112], while that of the latter, at least for the obligate hydrogenotrophic methanogens (see above) is still not known. However, since Mvh hydrogenase is tightly associated with HDR [137] it was proposed that electrons derived from H2 oxidation are transferred via Fe/S clusters and FAD from Mvh to both the heterodisulfide and ferredoxin by “electron bifurcation” [110]. M. voltae was shown to contain a Sec-dependent (Vhu and Fru) and a Sec-independent (Vhc and Frc) set of hydrogenases [138]. Of the former, two small subunits contain one (VhuU) and two Sec residues (VhuD), respectively, while the latter contains Sec in its large subunit (FruA) [139]. This situation appears to be identical in all Sec-encoding Methanococcus species for which genome sequences are available [125,126]. In M. voltae, regulation of the genes encoding Frc and Vhc (Cyscontaining) was shown to involve both selenium-dependent repres-

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sion and activation of transcription [140–142]. Repression is mediated by HrsM, a LysR-type regulator [143]. However, how the seleniumstatus is sensed in M. voltae and how this signal is transduced, is not known. 6.3. Formyl-methanofuran dehydrogenase FMD (Fig. 5) catalyzes the reduction of CO2 and methanofuran to formyl-methanofuran, which is the initial step in methanogenesis from CO2 in all methanogenic archaea. FMD is composed of five subunits of which two share considerable similarity with FDH, and contains either Mo or W, and Fe/S clusters [144,145]. A Sec-containing FMD was first reported for Methanopyrus kandleri [127] and genome sequence analysis shows that in all methanogens employing Sec at least one Sec-containing FMD subunit is encoded [125,126,146]. The same is true for HDR (see below). 6.4. Heterodisulfide reductase Methanosarcina species employ a (Sec-independent) membranebound, two-subunit, cytochrome b-containing HDR, which most probably accepts the electrons to form HS-CoM and HS-CoB from methanophenazine (a membrane-integral electron carrier functionally analogous to quinones) [147]. The reduction of methanophenazine by H2 is catalyzed by a Sec-independent, membrane-bound F420non-reducing hydrogenase [148]. Transfer of the electrons from H2 oxidation via methanophenazine and cytochrome b to the site of CoM-S-S-CoB reduction (HDR) leads to the generation of a proton motive force [149]. Obligate hydrogenotrophs like Methanococcus and Methanothermobacter species lack both cytochromes and methanophenazine. They reduce CoM-S-S-CoB by a cytoplasmic multienzyme complex composed of the F420-nonreducing hydrogenase (see above) and a soluble HDR (Fig. 5), an iron–sulfur (seleno-) flavoprotein. Potential flavin-dependent reduction of ferredoxin was proposed to proceed via “electron bifurcation” involving HDR [110]. Anyway, it is still unclear if the exergonic reduction of CoM-S-S-CoB is directly coupled to energy conservation by ion extrusion in these organisms [115]. The question why selenium utilization is so asymmetrically distributed among Archaea cannot be answered satisfactorily. As mentioned above, it is likely that selenium utilization is an original trait [150]; considering that most archaeal selenoproteins are involved in metabolism for which numerous special cofactors, and thus, the factors for their biogenesis, are required, it seems plausible that it is not easily distributed laterally; in fact, it appears that the Sec-utilizing trait was, and is being, lost in methanogens. Comparing the selenium requirement of M. jannaschii, M. voltae and M. maripaludis supports this scenario [89,118]. These organisms employ, to various degrees, isoforms where the Sec residue is replaced by Cys [118,127,130,138]. In M. voltae, for example, reduction of the selenium supply leads to reduced growth with H2 + CO2 [151], whereas in M. maripaludis, no effect upon selenium-deprivation is observed because the selenoproteins can be efficiently complemented by the corresponding Cysisoforms. Growth on formate, however, is impaired when selenium is scarce because formate dehydrogenase in this organism is strictly selenium-dependent [118]. In contrast, M. jannaschii only contains the Sec-dependent FDH, hydrogenase, and FMD. Its growth is therefore strictly dependent on the availability of selenium in the medium. 7. Selenoprotein synthesis in Archaea 7.1. Selenocysteine synthesis Investigating the path of Sec synthesis and incorporation in Archaea started with the available genome sequence of M. jannaschii and the finding that it produces selenoproteins [89,126]. The genome

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Fig. 6. Model of selenocysteine biosynthesis and incorporation in M. maripaludis and M. jannaschii. 3′UTR, 3′-untranslated region; PSTK, seryl-tRNAsec kinase, [Se], reduced Sespecies; SelB, Sec-specific elongation factor; SepSecS, O-phosphoseryl-tRNAsec:selenocysteine synthase; SerRS, seryl-tRNA synthetase; SPS, selenophosphate synthetase; see text for details.

was found to encode tRNAsec, which, in its predicted structure, resembled eukaryotic tRNAsec more than bacterial tRNAsec [21,116]. Archaeal tRNAsec could be overproduced in E. coli and, after purification, be selenocysteylated in vitro to Sec-tRNAsec with serine, ATP, selenide, and E. coli SerRS, SPS, and Sec synthase. Based on this finding a common mechanism for Sec synthesis in Bacteria and Archaea was assumed [152]. However, in neither Archaea nor Eukarya could a homolog of bacterial SelA been identified. A distant homolog from M. jannaschii was shown to have no Sec synthase activity [153]. Instead, an archaeal kinase was identified as potentially involved in Sec synthesis because homologs were only encoded in genomes also encoding Sec [154]. Biochemical characterization of a mammalian homolog showed that it catalyzes phosphorylation of the seryl-moiety of Ser-tRNAsec to O-phosphoseryl-tRNAsec leading to its designation as O-phosphoseryl-tRNAsec kinase (PSTK) [154]. The M. jannaschii homolog was found to catalyze the same reaction [153,155], but still the significance of this reaction in vivo was unclear. O-phosphoseryltRNA had been known in eukaryotes for decades [156] and it had been speculated that it could be an intermediate in Sec-tRNAsec formation or represent a “storage form” of Ser-tRNAsec [157]. This question could be solved when the coding sequence was determined for the antigen for autoantibodies leading to autoimmune hepatitis, known as soluble liver antigen/liver pancreas (SLA/LP) [158]. This information drew again attention of the selenium-community to SLA/LP because it was known to interact with eukaryal tRNAsec [159]. Bioinformatic analysis of the protein led to the proposal that SLA/LP might be the eukaryal Sec synthase due to its predicted PLP-dependence and its predicted overall similarity to bacterial Sec synthase [160]. Evidence for its involvement in eukaryal Sec-synthesis was gathered by showing that knock-down of the SLA/LP encoding mRNA leads to decreased levels of Sec incorporation in mammalian cells [161]. SLA/LP-specific autoimmune antisera interact with the M. jannaschii SLA/LP homolog [162] but its involvement in Sec-synthesis was finally proven by elegant heterologous genetic analysis [19]. A Sec synthase-deficient E. coli mutant regained its capacity to incorporate Sec only when the genes for both PSTK and SLA/LP, irrespective whether from an eukaryal or an archaeal source, were present [19]. In vitro characterization of the SLA/LP homolog of M. maripaludis demonstrated that it catalyzes the selenophosphate-dependent conversion of O-phosphoseryl-tRNAsec to Sec-tRNAsec [19]. Thus, the sequential action of PSTK, phosphorylating Ser-tRNAsec, and SLA/LP (now renamed O-phospho-

seryl-tRNAsec:selenocysteine synthase, SepSecS), converting the Ophosphoseryl-moiety to a selenocysteyl-moiety with selenophosphate as the selenium donor, was established (Fig. 6). An elaborate biochemical study, published simultaneously, showed that Sec synthesis in vitro proceeds via the identical pathway in eukaryotes [163] and the structures of both eukaryal (there called SecS), and archaeal SepSecS have been solved [164,165]. 7.2. Selenocysteine incorporation Analysis of Sec-encoding genes of M. voltae first showed that Sec insertion in Archaea is also directed by UGA [138]. However, Archaea (and Eukarya) do not contain conserved SECIS structures within the coding region of the selenoprotein mRNAs. Instead, conserved hairpin structures for different selenoprotein mRNAs of M. jannaschii could be found in the non-translated regions (UTRs) [89,116]. In six of the genes these elements are located close to the translational stop codon in the 3′-UTR; however, one appears to be located in the 5′-UTR [89]. The situation is very similar in M. maripaludis [125] and experimental proof for the nature of a putative 3′-UTR-hairpin structure as SECIS element was provided in vivo by showing that a selenoprotein gene from M. jannaschii could only be heterologously expressed in M. maripaludis in the presence of the SECIS element [128]; analysis of SECIS point mutants further showed that not the sequence, but rather the correct secondary and/or tertiary structure, is crucial for Sec insertion [128]. An interesting feature of Sec-decoding methanococci is that four of their selenoprotein genes are organized in a putative operon made of seven genes and that the four selenoprotein genes each have a SECIS element in the derived 3′ UTR of their mRNAs. This situation suggests that one SECIS serves in the translation of its immediate upstream reading frame; however, one of these genes (vhuD) encodes two Sec residues but only one 3′-UTR-SECIS suggesting that it functions in Sec insertion into more than one position, a situation known from eukaryal selenoprotein P [166,167] and only recently found in other prokaryotes [168]. In bacteria, the key component for the decoding of UGA with Sec is SelB (see above). A homologous protein from M. jannaschii was characterized in vitro and shown to display biochemical properties typical for SelB proteins [152]. Its in vivo role in archaeal selenoprotein synthesis was subsequently proven by mutational analysis. A M.

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maripaludis strain lacking SelB could not produce selenoproteins anymore but was still able to grow with H2 + CO2 as substrates [118], which was shown to be due to the capacity of the strain to produce a set of selenium-independent isoenzymes complementing for the selenoenzymes (Fig. 5). A striking difference to bacterial SelB is that archaeal SelB lacks parts of the C-terminal extension, which in bacterial SelB is responsible for SECIS binding [97,104]. Consequently, binding of the archaeal SelB to a cognate SECIS-element could not be demonstrated [152]. However, the potential role of aminoacyl-tRNAsec in SECIS-binding of SelB was not addressed in these studies and the crystal structure of SelB from M. maripaludis gave rise to speculations that it may bind the SECIS element [169,170]. In eukaryotes, SelB (also called eSelB or eEFsec) does not bind the SECIS element in vitro [171,172]. Instead, “SECIS-binding protein 2” (SBP2) binds the SECIS and stabilizes a SelB•SBP2•SECIS complex [172,173]. Furthermore, ribosomal protein L30 was also shown to bind eukaryal SECIS elements and to influence UGA recoding, which led to a model proposed for UGA decoding in eukaryotes [174] (and see Allmang et al., this issue). In Archaea, no SECIS-binding protein is known and no known archaeal genome encodes a SBP2 homolog; however, L30 homologs are encoded in many archaeal genomes. M. jannaschii, for example, encodes three proteins annotated as L30 [126]; one of them was tested whether it could function in eukaryal selenoprotein synthesis but failed [174]. Thus, the question as to how communication between the site of UGA recoding (the SECIS element) and the site of translation (SelB/ribosome) is established in Archaea, remains unanswered (Fig. 6). 8. Conclusion Despite the fact that the amount of sequence information through (meta-)genome sequencing projects is ever-increasing, and although comparative genome analyses and studies on archaeal selenoprotein synthesis have proved instrumental in elucidating the pathway of Sec synthesis in Archaea and Eukarya during the last years, there are still considerable gaps in our understanding of the biochemical and physiological function of selenoproteins, of the UGA decoding mechanism, and the evolution of the system for Sec synthesis and incorporation. For example, why is the Sec-decoding system of Grampositive bacteria so promiscuous? Can we use such a system to produce selenoproteins heterologously in E. coli on a routine basis? How is recoding of UGA to Sec brought about in Archaea? Is the twostep “archaeal/eukaryal way” of Sec synthesis from seryl-tRNAsec derived from the one-step “bacterial way” or vice versa, or were the two pathways invented independently? What is the selective advantage of spending an additional ATP in the two-step pathway? The fact that (i) the system for Sec synthesis and incorporation of M. maripaludis appears to be similar to the eukaryal one, (ii) the Sec-trait is dispensable in M. maripaludis, and (iii) the organism is amenable to straightforward and stable genetic manipulation holds the promise that it will be helpful to answer some of these questions in the future. Acknowledgements We are indebted to V. Müller, Frankfurt, for his support. M. R. wishes to express his gratitude to August Böck for his teachings. Work in the author's laboratory is supported by a grant from the Deutsche Forschungsgemeinschaft (through SFB 579). References [1] D.E. Hogue, SeleniumJ , Dairy Sci. 53 (1970) 1135–1137. [2] J. Pinsent, The need for selenite and molybdate in the formation of formic dehydrogenase by members of the coli–aerogenes group of bacteria, Biochem. J. 57 (1954) 10–16.

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