Thressa C. Stadtman

Selenium Biochemistry Proteins containing selenium are essential components of certain bacterial and mammalian enzyme systems

The toxicity of selenium to animals and plants has been known and extensively documented since the 1930s, but it is only during the past 15 years that selenium has also been shown to be an essential micronutrient for animals and bacteria. Very little is known about the specific role or roles of selenium and, to date, there are only three enzyme-catalyzed reactions that have been shown to require the participation of a selenium-containing protein. These are the reactions catalyzed by (i) formate dehydrogenase of bacteria, (ii) glycine reductase of clostridia, and (iii) glutathione peroxidase of erythrocytes. The common denominator of these selenium-dependent processes is that they are all oxidation-reduction reactions. A fourth selenoprotein has been isolated from skeletal muscle of sheep but its catalytic function has not been identified. The form in which selenium occurs in these selenoproteins is unknown. The selenoprotein of clostridial glycine reductase contains selenium in a covalently bound form. Studies in progress indicate that this may be an organoselenium compound not previously detected in nature. Identification of the chemical nature of selenium in proteins participating in electron transport processes should enable us to determine its specific role and to understand the basic defects in certain cardiac and skeletal muscle degenerative diseases which are selenium-deficiency syndromes. The greater availability and ease of isolation of the selenoprotein of the bacterial glycine reductase system makes this the biological material of choice for studies on the mechanism of action of selenium. An added attractive feature of this system is that it can conserve the energy made available by the reductive deamination of glycine in a biologically useful form by synthesizing ATP.

Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein

Abstract A procedure for the isolation in nearly homogeneous form of protein A, a low molecular-weight, acidic, protein component of clostridial glycine reductase, is described. The yield of protein A is high only in early log phase cells of Clostridium sticklandii grown under standard laboratory conditions in a rich tryptone-yeast extract-distilled water medium but, when selenite (1 μ m ) is added, the levels of protein A remain high throughout the entire log phase of growth. Addition of 75 Se-labeled selenite to the culture medium results in the highly selective incorporation of radioactive selenium into protein A. The procedure for isolation of protein A results in about a 700-fold enrichment when extracts prepared from cells that actively catalyze glycine reduction are used. However, the catalytic activity of the purified protein varies considerably from preparation to preparation. The molecular weight of protein A, estimated by sucrose density-gradient centrifugation, is approximately 12,000. The other higher molecular-weight components of glycine reductase are associated with the membrane fraction of the cell and are released as soluble proteins by sonic disruption of the membrane. After purification by ion-exchange and molecular sieve chromatography, these components are separated by DEAE-cellulose chromatography into two protein fractions both necessary for glycine reductase activity in protein A-supplemented assays. One of these fractions consists of a major protein component, protein B, also nearly homogeneous as determined by polyacrylamide gel electrophoresis. The other protein fraction still is heterogeneous.

Selenium Biochemistry: Mammalian Selenoenzymes

Within the past ten years selenium biochemistry has attracted an increasing number of investigators interested both in the beneficial and the toxic effects of the element in biological systems. In 1957 two independent research groups showed that the trace element selenium is an important nutrient for animals. Klaus Schwartz at the National Institutes of Health in Bethesda isolated a selenium-containing factor that prevented rats fed a Torula yeast based diet from developing liver necrosis. 1 Investigators at the Lederle Laboratories in Pearl River, NY, found that exudative diathesis in poultry was prevented by addition of selenium to the diet. 2 Predating these discoveries by three years was a report 3 that Escherichia coli required selenium and molybdenum for synthesis of active formate dehydrogenase, but this finding attracted little attention at a time when the revelance of discoveries made in bacterial physiology to mammalian physiology was not widely appreciated. A possible biochemical explanation of the beneficial effects of selenium in animals came several years later when the trace element was discovered to be an essential component of an important antioxidant enzyme, glutathione peroxidase. 4,5 At the same time a low molecular weight component, protein A, of the Clostridial glycine reductase complex was shown to be a selenoprotein 6 and the selenium-containing moiety was identified as selenocysteine. 7 The techniques developed for alkylation of the reduced selenocysteine and identification of the carboxymethyl and carboxyethyl derivatives of the selenoamino acid in acid hydrolysates of Clostridial selenoprotein A facilitated identification of selenocysteine in glutathione peroxidase and, later, in other selenoproteins. 8

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 2.3.1.1) 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\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{2-}}}\end{equation*}\end{document} directly, but in the presence of reduced glutathione (GSH) and SeO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{2-}}}\end{equation*}\end{document} E-Se rhodanese was generated. These results indicate that the intermediates produced from the reaction of GSH with SeO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{2-}}}\end{equation*}\end{document} 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°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.

Escherichia coli NifS-like Proteins Provide Selenium in the Pathway for the Biosynthesis of Selenophosphate

Selenophosphate synthetase (SPS), theselD 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 Se0-generating system consisting ofl-selenocysteine and the Azotobacter vinelandiiNifS protein can replace selenide for selenophosphate biosynthesisin 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 Se0 and S0, respectively. In the present study, we demonstrate the ability of each E. coliNifS-like protein to function as a selenium delivery protein for thein 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.

Occurrence of selenocysteine in the selenium-dependent formate dehydrogenase of Methanococcus vannielii.

The selenium-dependent formate dehydrogenase of Methanococcus vannielii was isolated from bacteria grown in the presence of [75Se]selenite. Purification under strictly anaerobic conditions resulted in the simultaneous enrichment of formate dehydrogenase activity, 75Se, and a brown chromophore that absorbs maximally at 380 nm. Acid hydrolysis of the enzyme after reduction with borohydride and alkylation with iodoacetamide, released a radioactive selenoamino acid derivative that was identified as [75Se]carboxymethyl-selenocysteine. This is the third selenoenzyme shown to contain selenocysteine.

The NIFS Protein Can Function as a Selenide Delivery Protein in the Biosynthesis of Selenophosphate

The NIFS protein from Azobacter vinelandii is a pyridoxal phosphate-containing homodimer that catalyzes the formation of equimolar amounts of elemental sulfur andl-alanine from the substrate l-cysteine (Zheng, L., White, R. H., Cash, V. L., Jack, R. F., and Dean, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2754–2758). A sulfur transfer role of NIFS in which the enzyme donates sulfur for iron sulfur center formation in nitrogenase was suggested. The fact that NIFS also can catalyze the decomposition ofl-selenocysteine to elemental selenium andl-alanine suggested the possibility that this enzyme might serve as a selenide delivery protein for the in vitrobiosynthesis of selenophosphate. In agreement with this hypothesis, we have shown that replacement of selenide with NIFS andl-selenocysteine in the in vitroselenophosphate synthetase assay results in an increased rate of formation of selenophosphate. These results thus support the view that a selenocysteine-specific enzyme similar to NIFS may be involved as anin vivo selenide delivery protein for selenophosphate biosynthesis. A kinetic characterization of the two NIFS catalyzed reactions carried out in the present study indicates that the enzyme favors l-cysteine as a substrate compared with its selenium analog. A specific activity for l-cysteine of 142 nmol/min/mg compared with 55 nmol/min/mg forl-selenocysteine was determined. This level of enzyme activity on the selenoamino acid substrate is adequate to deliver selenium to selenophosphate synthetase in the in vitroassay system described.

Direct detection of potential selenium delivery proteins by using an Escherichia coli strain unable to incorporate selenium from selenite into proteins.

Selenium can be metabolized for protein synthesis by two major pathways in vivo. In a specific pathway it can be inserted into polypeptide chains as the amino acid selenocysteine, as directed by the UGA codon. Alternatively, selenium can be substituted for sulfur to generate the free amino acids selenocysteine and selenomethionine, and these are incorporated nonspecifically into proteins in place of cysteine and methionine, respectively. A mutant strain of Escherichia coli was constructed that is deficient in utilization of inorganic selenium for both specific and nonspecific pathways of selenoprotein synthesis. Disruption of the cysK gene prevented synthesis of free cysteine and selenocysteine from inorganic S and Se precursors. Inactivation of the selD gene prevented synthesis of selenophosphate, the reactive selenium donor, required for the specific incorporation pathway. As expected, the double mutant strain, RL165ΔselD, when grown anaerobically in LB + glucose medium containing 75SeO32−, failed to synthesize selenium-dependent formate dehydrogenase H and seleno-tRNAs. However, it incorporated 24% as much selenium as the wild-type strain. Selenium in the deficient strain was bound to five different proteins. A 39-kDa species was identified as glyceraldehyde-3-phosphate dehydrogenase. It is possible that selenium was bound as a perselenide derivative to the reactive cysteine residue of this enzyme. A 28-kDa protein identified as deoxyribose phosphate aldolase also contained bound selenium. These 75Se-labeled proteins may have alternate roles as selenium delivery proteins.

Methane biosynthesis by Methanosarcina barkeri: Properties of the soluble enzyme system

Abstract Extracts of Methanosarcina barkeri reduce the following substrates to methane: methanol, the methyl moiety of methylcobalamin, methyltetrahydrofolate, formaldehyde, formate, the carboxyl carbon of pyruvate, and carbon dioxide. Crude extracts utilize either molecular hydrogen or pyruvate as electron donor; in addition ATP and, when pyruvate is electron donor, coenzyme A are required. A role of a vitamin B12 type of compound in all these reductions is suggested by the potent inhibitory effects exerted by intrinsic factor. When substrate levels of B12s are added as a trapping agent, M. barkeri extracts convert methanol to methylcobalamin which accumulates in the reaction mixture. This reaction also is dependent on ATP.

The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H

Three NifS-like proteins, IscS, CSD, and CsdB, from Escherichia coli catalyze the removal of sulfur and selenium from l-cysteine and l-selenocysteine, respectively, to form l-alanine. These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin. Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNA in vivo, supporting the involvement of the NifS-like proteins in selenium metabolism. We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm5s2U) in tRNA, suggesting that the sulfur atom released from l-cysteine by the action of IscS is incorporated into mnm5s2U. In contrast, neither CSD nor CsdB was essential for production of mnm5s2U and 5-methylaminomethyl-2-selenouridine. The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H. Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.