Gerard M. Lacourciere

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.

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.

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.

Selenium is mobilized in vivo from free selenocysteine and is incorporated specifically into formate dehydrogenase H and tRNA nucleosides.

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. It was recently demonstrated that selenium delivered from selenocysteine by an E. coli NifS-like protein could replace free selenide in the in vitro SPS assay for selenophosphate formation (G. M. Lacourciere, H. Mihara, T. Kurihara, N. Esaki, and T. C. Stadtman, J. Biol. Chem. 275:23769-23773, 2000). During growth of E. coli in the presence of 0.1 microM (75)SeO(3)(2-) and increasing amounts of L-selenocysteine, a concomitant decrease in (75)Se incorporation into formate dehydrogenase H and nucleosides of bulk tRNA was observed. This is consistent with the mobilization of selenium from L-selenocysteine in vivo and its use in selenophosphate formation. The ability of E. coli to utilize selenocysteine as a selenium source for selenophosphate biosynthesis in vivo supports the participation of the NifS-like proteins in selenium metabolism.

Selenophosphate — selenium donor for protein and tRNA

Monoselenophosphate has been identified as the activated selenium donor in Escherichia coli that is required for both the biosynthesis of selenocysteine in selenium-dependent enzymes and for the conversion of 2-thiouridine residues to 2-selenouridine in tRNA. Selenophosphate is generated from ATP and selenide by the selD gene product, selenophosphate synthetase.