Cloning and heterologous expression of a Methanococcus vannielii gene encoding a selenium-binding protein.

The activation and incorporation of selenium into selenocysteine containing selenoproteins has been well established in an Escherichia coli model system but there is little specific information concerning the transport and intracellular trafficking of selenium in biological systems in general. A selenium transport role is a possible function of a novel 42 kDa selenium-binding protein that recently was purified from Methanococcus vannielii. The gene encoding a monomer of this protein (Sbp) has been cloned, sequenced and heterologously expressed in E. coli. The 8.8 kDa gene product contains 81 amino acids. The recombinant Sbp (rSbp) protein was shown to bind selenium from added selenite. The bound selenium appeared predominantly in dimeric and tetrameric forms of the protein. The gene encoding Sbp occurs in an operon that contains a carbonic anhydrase gene and selenocysteine-containing formate dehydrogenase genes, suggesting possible roles in selenium-dependent formate metabolism.

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis.

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

Formation of a selenium-substituted rhodanese by reaction with selenite and glutathione: possible role of a protein perselenide in a selenium delivery system.

Selenophosphate is the active selenium-donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes. Although free selenide can be used in vitro for the synthesis of selenophosphate, the actual physiological selenium substrate has not been identified. Rhodanese (EC ) normally occurs as a persulfide of a critical cysteine residue and is believed to function as a sulfur-delivery protein. Also, it has been demonstrated that a selenium-substituted rhodanese (E-Se form) can exist in vitro. In this study, we have prepared and characterized an E-Se rhodanese. Persulfide-free bovine-liver rhodanese (E form) did not react with SeO(3)(2-) directly, but in the presence of reduced glutathione (GSH) and SeO(3)(2-) E-Se rhodanese was generated. These results indicate that the intermediates produced from the reaction of GSH with SeO(3)(2-) are required for the formation of a selenium-substituted rhodanese. E-Se rhodanese was stable in the presence of excess GSH at neutral pH at 37 degrees C. E-Se rhodanese could effectively replace the high concentrations of selenide normally used in the selenophosphate synthetase in vitro assay in which the selenium-dependent hydrolysis of ATP is measured. These results show that a selenium-bound rhodanese could be used as the selenium donor in the in vitro selenophosphate synthetase assay.

Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity.

In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.

Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate.

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.

Selenium-dependent metabolism of purines: A selenium-dependent purine hydroxylase and xanthine dehydrogenase were purified from Clostridium purinolyticum and characterized.

During purification of the selenium-dependent xanthine dehydrogenase (XDH) from Clostridium purinolyticum, another hydroxylase was uncovered that also contained selenium and exhibited similar spectral properties. This enzyme was purified to homogeneity. It uses purine, 2OH-purine, and hypoxanthine as substrates, and based on its substrate specificity, this selenoenzyme is termed purine hydroxylase (PH). The product of hydroxylation of purine by PH is xanthine. A concomitant release of selenium from the enzyme and loss of catalytic activity on treatment with cyanide indicates that selenium is essential for PH activity. Selenium-dependent XDH, also purified from C. purinolyticum, was found to be insensitive to oxygen during purification and to use both potassium ferricyanide and 2,6-dichloroindophenol as electron acceptors. Selenium is required for the xanthine-dependent reduction of 2, 6-dichloroindophenol by XDH. Kinetic analyses of both enzymes revealed that xanthine is the preferred substrate for XDH and purine and hypoxanthine are preferred by PH. This characterization of these selenium-requiring hydroxylases involved in the interconversion of purines describes an extension of the pathway for purine fermentation in the purinolytic clostridia.

Mammalian thioredoxin reductase: oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity.

Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys(59)/Cys(64) adjacent to the flavin ring of FAD and another center consisting of Cys(497)/selenocysteine (SeCys)(498) near the C terminus. We now show that the C-terminal Cys(497)-SH/SeCys(498)-Se(-) of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS(2)) or H(2)O(2). The Cys(59)-SH/Cys(64)-SH pair also was oxidized to a disulfide. At lower concentrations of TrxS(2), the Cys(59)-SH/Cys(64)-SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys(497)-SH/SeCys(498)-Se(-). Specific alkylation of SeCys(498) completely blocked the TrxS(2)-induced oxidation of Cys(59)-SH/Cys(64)-SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys(498) with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli. The NADPH-disulfide oxidoreductase activity of the purified Cys(497)/Cys(498) mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5, 5'-dithiobis(2-nitrobenzoic acid) or TrxS(2), respectively, as substrate. Disulfide formation induced by excess TrxS(2) in the mutant form was 12% of that of the wild type. Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.

Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities.

The TrxRl form of thioredoxin reductase (TrxR) was the major form of the enzyme isolated from HeLa cells grown in a fermentor at 35 degrees C under controlled aeration conditions favorable to growth, nominally 30% of saturation of dissolved oxygen or 8 ml of oxygen in a liter of medium. This TrxR1 form was not retained on a heparin affinity matrix, it contained one selenium per subunit, was highly active catalytically, and showed strong cross-reactivity with anti-rat liver TrxR1 polyclonal antibodies. At higher aeration, 50% of saturation of dissolved oxygen or 12 ml of oxygen in a liter of medium, HeLa cell growth was slower and additional TrxR forms that bound to heparin were present in purified enzyme preparations. A minor component, TrxR2, the mitochondrial form of TrxR, was detected in the heparin-bound enzyme fraction. One enzyme form that contained less selenium (ca. 0.5 Se per TrxR subunit) was only about 50% as active with thioredoxin or 5,5'dithiobis(2-nitrobenzoic acid) as substrate. Cross-reactivity of this form with anti-rat liver TrxR1 polyclonal antibodies was very weak. The isoelectric point of the low Se enzyme, 5.85, was higher than that, 5.2-5.4, of normal Se content enzyme. Affinity of purified fully active TrxR1 to heparin could be induced by reduction with NADPH or tris-(2-carboxyethyl)phosphine (TCEP). Under anaerobic conditions there was complete retention of Se indicating that an enzyme conformation change effected by reduction was involved. The TCEP-reduced enzyme form was very oxygen labile and upon exposure to air both the Se content and catalytic activity decreased by about 50%. Addition of millimolar concentrations of NADPH or NADP(+) to the TCEP-reduced enzyme gave full protection from oxygen inactivation. TrxR1 exhibited weak peroxidase activity with H(2)O(2) as substrate in the presence of an NADPH-generating system but this activity was unstable. Specific alkylation of the selenocysteine residue of TrxR1 which completely inhibits the NADPH-dependent reduction of disulfides also destroyed peroxidase activity.

Responsiveness of selenoproteins to dietary selenium.

Selenocysteine-containing enzymes that have been identified in mammals include the glutathione peroxidase family (GPX1, GPX2, GPX3, and GPX4), one or more iodothyronine deiodinases and two thioredixin reductases. Selenoprotein P, a glycoprotein that contains 10 selenocysteine residues per 43 kDa polypeptide and selenoprotein W, a 10 kDa muscle protein, are unidentified as to function. Levels of all of these selenocysteine-containing proteins in various tissues are affected to different extents by selenium availability. Increased amounts of selenoproteins observed in response to selenium supplementation were shown in several studies to correlate with increases in the corresponding mRNA levels. In general, selenoprotein levels in brain are less sensitive to dietary selenium fluctuation than the corresponding selenoprotein levels in other tissues.

Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer.

Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a molybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of 13C-labeled formate in 18O-enriched water catalyzed by FDH(Se) produces 13CO2 gas that contains no 18O-label, establishing that the enzyme is not a member of the large class of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is coordinated in the reduced Mo(IV) state in a square pyramidal geometry to the four equatorial dithiolene sulfur atoms from a pair of pterin cofactors and a Se atom of the selenocysteine-140 residue [Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., and Sun, P. D. (1997) Science 275, 1305-1308]. EPR spectroscopy of the Mo(V) state indicates a square pyramidal geometry analogous to that of the Mo(IV) center. The strongest ligand field component is likely the single axial Se atom producing a ground orbital configuration Mo(dxy). The Mo-Se bond was estimated to be covalent to the extent of 17-27% of the unpaired electron spin density residing in the valence 4s and 4p selenium orbitals, based on comparison of the scalar and dipolar hyperfine components to atomic 77Se. Two electron oxidation of formate by the Mo(VI) state converts Mo to the reduced Mo(IV) state with the formate proton, Hf+, transferring to a nearby base Y-. Transfer of one electron to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) state. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar 1Hf hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the 1Hf hyperfine splitting from YHf, suggesting photoisomerizaton of this group or phototransfer of the proton to a more distant proton acceptor group A-. The minor effect of photolysis on the 77Se-hyperfine interaction with [77Se] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is located in the putative substrate-binding pocket close to the [Mo-Se] center. We propose that the transfer of Hf+ from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation of the formate molecule, thereby facilitating formation of CO2. Under normal physiological conditions, when electron flow is not limited by the terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and transfer of Hf+ against the thermodynamic potential.

Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism.

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.

Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells.

Mammalian selenocysteine-containing thioredoxin reductase (TR) isolated from HeLa cells and from human lung adenocarcinoma cells was separated into two major enzyme species by heparin-agarose affinity chromatography. The low-affinity enzyme forms that were not retained on heparin agarose showed strong crossreactivity in immunoblot assays with anti-rat liver TR polyclonal antibodies, whereas the high-affinity enzyme forms that were retained by the heparin column were not detected. Both low and high heparin-affinity enzyme forms contained FAD, were indistinguishable on SDS/PAGE analysis, and exhibited similar catalytic activities in the NADPH-dependent DTNB [5,5'-dithiobis(2-nitrobenzoate)] assay. The C-terminal amino acid sequences of 75Se-labeled tryptic peptides from lung adenocarcinoma low- and high heparin-affinity enzyme forms were identical to the predicted C-terminal sequence of human placental TR. These two determined peptide sequences were -Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Secys-(Gly). Occurrence of the Se-carboxymethyl derivative of radioactive selenocysteine in the position corresponding to TGA in the gene confirmed that UGA is translated as selenocysteine. The presence of cysteine followed by a reactive selenocysteine residue in this C-terminal region of the protein may explain some of the unusual properties of the mammalian TRs.

Selenophosphate synthetase: enzyme labeling studies with [gamma-32P]ATP, [beta-32P]ATP, [8-14C]ATP, and [75Se]selenide.

Selenophosphate synthetase catalyzes a reaction in which ATP and selenide are converted to H3SeP03, H3P04, and AMP in a 1:1:1 ratio. Selenophosphate is derived from the gamma phosphoryl group and orthophosphate from the beta phosphoryl group of ATP. In the absence of selenide, a slow reaction in which ATP is converted quantitatively to 2 H3P04 and AMP occurs. Labeling experiments carried out to detect a putative enzyme-bound pyrophosphate intermediate in the overall reaction showed that up to 0.6 equivalent of the 32P label from [gamma-32P]ATP was bound to protein under enzyme turnover conditions, but only a negligible amount of 32P from [beta-32P]ATP was present. Thus, no Enz-PP intermediate was present in a detectable amount under the experimental conditions used. Isolated enzyme samples contained 75Se from 75Se-labeled selenide and [14C]AMP from [8-14C]ATP in amounts similar to the bound 32P from [gamma-32P]ATP, suggesting that two of the final products, selenophosphate and AMP, were the radioactive compounds detected in these experiments.

A non-radioactive and two radioactive assays for selenophosphate synthetase activity.

Selenophosphate synthetase catalyzes the formation of monoselenophosphate (SePO3(3-)) from ATP and selenide (reaction 1). [formula: see text] In one assay frequently used, [8-14C]AMP formation from [8-14C]ATP is estimated after separation of the nucleotides by thinlayer chromatography. An alternative non-radioactive assay in which the AMP product is estimated using AMP deaminase is described. The highly oxygen-labile selenophosphate product can be estimated in an assay employing [gamma-32P]ATP. The 32P-labeled selenophosphate is converted to [32P]orthophosphate by treatment with iodine and estimated after removal of residual [32P]ATP on charcoal.

Characterization of crystalline formate dehydrogenase H from Escherichia coli. Stabilization, EPR spectroscopy, and preliminary crystallographic analysis.

The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.

A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity.

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri.

NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of higher specific activity then previously reported. NAH is most stable at alkaline pH in the presence of glycerol. The protein which consists of four dissimilar subunits occurs in forms of different molecular masses. There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein promoter. Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium. Mo(V), flavin radical, and two Fe2S2 clusters could be observed with EPR spectroscopy. The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released from NAH as a reactive low molecular weight compound by a number of denaturing procedures. Parallel losses of Se and catalytic activity were observed during purification and storage of the enzyme. Addition of sodium selenide or selenophosphate did not restore the catalytic activity of the enzyme. Instead, NAH is reversibly inactivated by these compounds and also by sulfide. Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity. The "as isolated" enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detected at early stages of purification. NAH exhibits a high substrate specificity toward electron donor substrates. The ability of a nicotinate analog to reduce NAH (disappearance of 2.067 signal) correlates with the rate of oxidation of the analog in the standard assay mixture. The properties of NAH differentiate the enzyme from known Mo-containing hydroxylases.

Selenocysteine.

Selenocysteine is recognized as the 21st amino acid in ribosome-mediated protein synthesis and its specific incorporation is directed by the UGA codon. Unique tRNAs that have complementary UCA anticodons are aminoacylated with serine, the seryl-tRNA is converted to selenocysteyl-tRNA and the latter binds specifically to a special elongation factor and is delivered to the ribosome. Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine. A reactive oxygen-labile compound, selenophosphate, is the selenium donor required for synthesis of selenocysteyl-tRNA. Selenophosphate synthetase, which forms selenophosphate from selenide and ATP, is found in various prokaryotes, eukaryotes, and archaebacteria. The distribution and properties of selenocysteine-containing enzymes and proteins that have been discovered to date are discussed. Artificial selenoenzymes such as selenosubtilisin have been produced by chemical modification. Genetic engineering techniques also have been used to replace cysteine residues in proteins with selenocysteine. The mechanistic roles of selenocysteine residues in the glutathione peroxidase family of enzymes, the 5' deiodinases, formate dehydrogenases, glycine reductase, and a few hydrogenases are discussed. In some cases a marked decrease in catalytic activity of an enzyme is observed when a selenocysteine residue is replaced with cysteine. This substitution caused complete loss of glycine reductase selenoprotein A activity.

Glycine reductase selenoprotein A is not a glycoprotein: the positive periodic acid-Schiff reagent test is the result of peptide bond cleavage and carbonyl group generation.

The complete amino acid sequence of Clostridium sticklandii selenoprotein A, a selenocysteine-containing protein component of the glycine reductase complex, has been established. Both the intact protein and peptide fragments produced by Staphylococcus aureus V8 protease or trypsin were purified by reversed-phase high-performance liquid chromatography and subjected to electrospray ionization mass spectrometric analysis and standard Edman degradation. Selenoprotein A consists of 157 amino acids with a chemical molecular weight of 17,011, in reasonable agreement with the observed molecular weight (17,022.7) determined from its ionization mass spectrum. The sequence of the amino-terminal region of the isolated native protein is Ser-Arg-Phe-Thr-Gly-Lys- Lys-Ile-Val-Ile-Ile-Gly-Asp-Arg-Asp-. An N-terminal methionine residue deduced from the gene sequence was not present. Although selenoprotein A reacted positively in a glycoprotein stain when using either the periodic acid-Schiff reagent procedure or a commercial glycan detection kit, no saccharide was detected by carbohydrate analyses after acid hydrolysis or methanolysis. Identity of the amino acid sequence determined by analysis with that deduced from the gene sequence is further evidence of the absence of bound carbohydrate.