Selenocysteine synthase from Escherichia coli. Analysis of the reaction sequence

The product of the selA gene, selenocysteine synthase, is a pyridoxal 5-phosphate-containing enzyme which catalyzes the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA). Reduction of the aldimine group of pyridoxal 5-phosphate inactivates the enzyme. When reacted with seryl-tRNA(Sec UCA) as sole substrate, pyruvate (and possibly also ammonia) is released; in the presence of a high concentration of potassium borohydride, alanyl-tRNA(Sec UCA) is formed from seryl-tRNA(Sec UCA). These results support the notion that the formyl group of pyridoxal phosphate forms a Schiff base with the alpha-amino group of L-serine with the subsequent 2,3-elimination of a water molecule and the generation of an aminoacrylyl-tRNA(Sec UCA) intermediate. ATP is not required for this reaction step, but it is necessary for the conversion of aminoacrylyl-tRNA into selenocysteyl-tRNA(Sec UCA) which, in addition, requires the SELD protein and reduced selenium. Selenocysteine synthase forms a stable complex with seryl-tRNA(Sec UCA) with one tRNA molecule bound per two 50-kDa monomers. The enzyme does not interact with serine-inserting tRNA species. Taken together, the results show that biosynthesis of selenocysteine takes place in the enzyme-bound state and involves the dehydration of L-serine esterified to tRNA in a first step formally followed by the 2,3-addition of HSe- which is provided by the SELD protein in an ATP-dependent reaction in the form of a reactive selenium donor molecule.     

The function of selenocysteine synthase and SELB in the synthesis and incorporation of selenocysteine.

The selAB operon codes for the proteins selenocysteine synthase and SELB which catalyse the synthesis and cotranslational insertion of selenocysteine into protein. This communication deals with the biochemical characterisation of these proteins and in particular with their specific interaction with the selenocysteine-incorporating tRNA(Sec). Selenocysteine synthase catalyses the synthesis of selenocysteyl-tRNA(Sec) from seryl-tRNA(Sec) in a pyridoxal phosphate-dependent reaction mechanism. The enzyme specifically recognizes the tRNA(Sec) molecule; a cooperative interaction between the tRNA binding site and the catalytically active pyridoxal phosphate site is suggested. SELB is an EF-Tu-like protein which specifically complexes selenocysteyl-tRNA(Sec). Interaction with the selenol group of the side chain of the aminoacylated residue is a prerequisite for the formation of a stable SELB.tRNA complex. Mechanistically, this provides the biochemical basis for the exclusive selection of selenocysteyl-tRNA(Sec) in the decoding step of a selenocysteine-specific UGA triplet.     

Structural and functional investigation of a putative archaeal selenocysteine synthase

Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.     

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.     

Selenocysteine synthase from Escherichia coli. Nucleotide sequence of the gene (selA) and purification of the protein

The nucleotide sequence of the selA gene from Escherichia coli whose product is involved in the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA) was determined. selA codes for a polypeptide of a calculated Mr of 50,667; a protein of appropriate size was synthesized in vivo in a T7 promoter/polymerase system. An assay for SELA activity was devised which is based on the seryl-tRNA(Sec UCA)-dependent incorporation of [75Se] selenium into acid-insoluble material. It was used to follow SELA purification from cells that overproduced the protein from a phage T7 promoter plasmid. Purified native SELA protein migrates in gel filtration experiments with a native Mr of about 600,000. SELA contains 1 mol of bound pyridoxal 5-phosphate/mol of 50-kDa subunit. Evidence is presented that the overall conversion of seryl-tRNA(Sec UCA) to selenocysteyl-tRNA(Sec UCA) occurs at the SELA protein. SELA, therefore, has the function of a selenocysteine synthase.     

Characterization of the SECIS binding protein 2 complex required for the co-translational insertion of selenocysteine in mammals

Selenocysteine is incorporated into at least 25 human proteins by a complex mechanism that is a unique modification of canonical translation elongation. Selenocysteine incorporation requires the concerted action of a kink-turn structural RNA (SECIS) element in the 3′ untranslated region of each selenoprotein mRNA, a selenocysteine-specific translation elongation factor (eEFSec) and a SECIS binding protein (SBP2). Here, we analyze the molecular context in which SBP2 functions. Contrary to previous findings, a combination of gel filtration chromatography and co-purification studies demonstrates that SBP2 does not self-associate. However, SBP2 is found to be quantitatively associated with ribosomes. Interestingly, a wild-type but not mutant SECIS element is able to effectively compete with the SBP2 ribosome interaction, indicating that SBP2 cannot simultaneously interact with the ribosome and the SECIS element. This data also supports the hypothesis that SBP2 interacts with one or more kink turns on 28S rRNA. Based on these results, we propose a revised model for selenocysteine incorporation where SBP2 remains ribosome bound except during selenocysteine delivery to the ribosomal A-site.     

The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu

Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc] or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and B?ck, A. (1990) Nucleic Acids Res. 18, 6761-6766). tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu. It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser). tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec). Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec). Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly. Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine. The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.     

Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis

Selenocysteine (Sec), the 21^st amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNA^Sec). In archaea and eukaryotes, O-phosphoseryl-tRNA^Sec:selenocysteinyl-tRNA^Sec synthase (SepSecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while being attached to tRNA^Sec. We have previously shown that only the SepSecS tetramer is capable of binding to and recognizing the distinct fold of tRNA^Sec. Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised regarding whether the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small angle x-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between SepSecS and tRNA^Sec in vivo. We show that SepSecS preferentially binds one or two tRNA^Sec molecules at a time and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between SepSecS and two tRNAs, one enzyme homodimer plays a role of the noncatalytic unit that positions CCA ends of two tRNA^Sec molecules into the active site grooves of the other, catalytic, homodimer. Finally, our results demonstrate that the previously determined crystal structure represents the physiologically and catalytically relevant complex and suggest that allosteric regulation of SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

Microbial distribution of selenocysteine lyase.

We studied the distribution of selenocysteine lyase, a novel enzyme catalyzing the conversion of selenocysteine into alanine and H2Se, which we first demonstrated in various mammalian tissues (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). Enzyme activity was found in various bacteria such as Alcaligenes viscolactis and Pseudomonas alkanolytica. No significant activity was found in yeasts and fungi. Selenocysteine lyases from A. viscolactis and P. alkanolytica acted specifically on L-selenocysteine and required pyridoxal 5'-phosphate as a cofactor.     

Distinct genetic code expansion strategies for selenocysteine and pyrrolysine are reflected in different aminoacyl-tRNA formation systems

Selenocysteine and pyrrolysine, known as the 21st and 22nd amino acids, are directly inserted into growing polypeptides during translation. Selenocysteine is synthesized via a tRNA-dependent pathway and decodes UGA (opal) codons. The incorporation of selenocysteine requires the concerted action of specific RNA and protein elements. In contrast, pyrrolysine is ligated directly to tRNA^Pyl and inserted into proteins in response to UAG (amber) codons without the need for complex re-coding machinery. Here we review the latest updates on the structure and mechanisms of molecules involved in Sec-tRNA^Sec and Pyl-tRNA^Pyl formation as well as the distribution of the Pyl-decoding trait.     

Processive Selenocysteine Incorporation during Synthesis of Eukaryotic Selenoproteins

Selenoproteins are a family of proteins that share the common feature of containing selenocysteine, the “twenty-first” amino acid. Selenocysteine incorporation occurs during translation of selenoprotein messages by redefinition of UGA codons, which normally specify termination of translation. Studies of the eukaryotic selenocysteine incorporation mechanism suggest that selenocysteine insertion is inefficient compared with termination. Nevertheless, selenoprotein P and several other selenoproteins are known to contain multiple selenocysteines. The production of full-length (FL) protein from these messages would seem to demand highly efficient selenocysteine incorporation due to the compounding effect of termination at each UGA codon. We present data demonstrating that efficient incorporation of multiple selenocysteines can be reconstituted in rabbit reticulocyte lysate translation reactions. Selenocysteine incorporation at the first UGA codon is inefficient but increases by approximately 10-fold at subsequent downstream UGA codons. We found that ribosomes in the “processive” phase of selenocysteine incorporation (i.e., after decoding the first UGA codon as selenocysteine) are fully competent to terminate translation at UAG and UAA codons, that ribosomes become less efficient at selenocysteine incorporation as the distance between UGA codons is increased, and that efficient selenocysteine incorporation is not dependent on cis-acting elements unique to selenoprotein P. Furthermore, we found that the percentage of ribosomes decoding a UGA codon as selenocysteine rather than termination can be increased by 3- to 5-fold by placing the murine leukemia virus UAG read-through element upstream of the first UGA codon or by providing a competing messenger RNA in trans. The mechanisms of selenocysteine incorporation and selenoprotein synthesis are discussed in light of these results.     

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).     

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman’s reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.     

Dissociation of cAMP changes and myocardial contractility in taurine perfused rat heart.

Selenocysteine in the catalytic site of glutathione peroxidase was stabilized by conversion to the carboxymethyl derivative. A selenium-containing tryptic fragment was partially purified by column chromatography through cellulose phosphate, Sephadex G-25 superfine, DEAE-Agarose, and again through Sephadex G-25 superfine. Automated sequential Edman degradation yielded a residue of the phenylthiohydantoin of carboxymethyl-selenocysteine, indicating that the selenocysteine in the native enzyme is located within the polypeptide chain.     

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.     

Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism

The opal termination codon UGA is used in both prokaryotic and eukaryotic species to direct the specific insertion of selenocysteine into certain selenium-dependent enzymes. So far a formate dehydrogenase (hydrogenase-linked) of Escherichia coli and glutathione peroxidases of murine, human and rat origin have been identified as enzymes containing selenocysteine residues encoded by UGA. A novel seryl-tRNA, anticodon UCA, that specifically recognizes the UGA codon is required for selenocysteine incorporation into formate dehydrogenase. A eukaryotic UGA suppressor tRNA with UCA anticodon that accepts serine and is phosphorylated to O-phosphoseryl-tRNA may have a corresponding function in glutathione peroxidase synthesis. Other factors required for the unusual usage of the in-frame UGA codons to specify selenocysteine incorporation and the biochemical mechanism involved in distinguishing these from normal UGA termination codons are discussed.