Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety.

Abstract

An acidic, heat-stable protein of molecular weight about 12,000 is one of the components of the enzyme system that catalyzes the reductive deamination of glycine (1, 2). The biological activity of this protein depends on the presence of 1 g-atom of covalently bound selenium per mol of protein (2, 3). Selenium also is known to be essential for the biological activity of mammalian (4) and avian (5) glutathione peroxidase and formate dehydrogenase of Escherichia coli (6-8) and various anaerobic bacteria (3, 9, 10). The selenium-containing moieties of these two enzymes have not been identified. In this communication evidence is presented that the organoselenium moiety of the clostridial glycine reductase selenoprotein is a selenocysteine residue. We have thus identified an essential selenium-containing residue in a protein. MATERIALS AND METHODSThe selenoprotein of the glycine reductase system was isolated from sonic extracts of Clostridium sticklandii (1, 2). Chromatography on Affi-Gel 501, a mercuribenzoate-agarose preparation supplied by Bio-Rad Laboratories, was used as a final isolation step for some preparations that were otherwise difficult to free of the last traces of impurities. * Cells grown in the presence of 1 ,uM Na275SeO3 and 2 mM Na2S were the source of the 75Se-labeled protein (2). The yeast extract and tryptone in the culture medium supplied additional sulfur, and the resultant high ratio of sulfur to selenium suppressed nonspecific substitution of the latter for sulfur in the proteins and other constituents of the cell.Other reagents were purchased as follows: carboxypeptidase Selenocystine was reduced to selenocysteine with KBH4, and the Se-carboxamidomethyl, Se-carboxymethyl, Se-carboxyethyl, and Se-aminoethyl derivatives of the selenol were prepared by procedures similar to those used for synthesis of the corresponding S-alkyl derivatives of cysteine (11). On cellulose thin-layer sheets these derivatives migrated with the following RF values: in 2-propanol/HCOOH/H20 (60:3:15) Se-carboxamidomethylselenocysteine, 0.18 and Se-carboxymethylselenocysteine, 0.35; in tertiary butyl alcohol/methylethylketone/88% HCOOH/H20 (40:30:15:15) Se-carboxymethylselenocysteine, 0.47, Se-carboxyethylselenocysteine, 0.50, and selenocystine, 0.1 in 1-propanol/5% NH40H (70:30) Se-carboxymethylselenocysteine, 0.44; in CHC13/CH30H/15% NH40H (40:40:10) Se-aminoethylselenocysteine, 0.46; and in 2-propanol/28% NH4OH/H20 (60:1.5:30) Se-aminoethylselenocysteine, 0.79, and selenocystine, 0.72. In each instance the RF of the corresponding sulfur compound was similar. RESULTSThe unusual absorption spectrum of the selenoprotein of glycine reductase (Fig. 1) is due to its high content of phenylalanine relative to tyrosine and to the absence of tryptophan. Almost identical spectra are exhibited by the parvalbumin of dogfish (12) and the calcium-binding protein component of troponin from rabbit muscle (13). The spectrum of the latter, like that of the selenoprotein, exhibits a shoulder in the 295-to 320-nm region. In neither insta...

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

“…These experiments were performed anaerobically because the selenocysteine residue is oxygen labile (Cone et al 1976). hnportantly, the SELB-GTP-SectRNA^'^'' temary complex gave the same toeprint observed with SELB alone and did not toeprint the G25C mutant.…”

“…Selenium has been identified as a covalent base modification in tRNA from bacteria as well as mammals (Chen and Stadtman 1980;Ching and Stadtman 1982;Ching 1984;Wittwer et al 1984;Wittwer and Ching 1989;Kramer and Ames 1988;Ching 1984) and is incorporated into protein cotranslationally as the amino acid selenocysteine (Cone et al 1976;Forstrom et al 1978;Stadtman 1990;Bock et al 1991). Selenoproteins typically contain one selenocysteine moiety per peptide chain although the mammalian plasma protein, selenoprotein P, has been reported to have as many as 10 selenocysteine residues (Hill et al 1991(Hill et al , 1993.…”

Selenium in Biology: Facts and Medical Perspectives

“…Selenium usually exerts its influence on physiology as an integral component of proteins, into which it is incorporated in the form of selenocysteine (Cone et al, 1976;see below). Selenoproteins can be selectively labeled by 75 Se in selenium-deficient animals and autoradiographically visualized after electrophoretic separation (Behne et al, 1996).…”

Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii

“…The solubilised glycine reductase utilises artificial electron donors such as methyl red viologen or dithiothreitol as the reductant [2] thus by-passing some of the components of the electron transport system which in vivo may interact with NAD(P)H. The complex has been further fractionated into a highly acidic, heat-stable protein 'A' ( M , 12000), containing a functionally essential selenocysteine residue [3,4], which has been purified to homogeneity [ 5 ] . The remaining fraction required for the reductive deamination of glycine is composed of at least three proteins which are in the process of being separated and purified in the laboratory of Stadtman [3,15].…”