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T C Stadtman

Publications and source records attributed to T C Stadtman.

At least 19 recordsLinked to original sources
PubMed1992

Escherichia coli mutant SELD enzymes. The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide.

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Böck, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.

Adenosine Triphosphate↗
PubMed1992

Synthesis of 5-methylaminomethyl-2-selenouridine in tRNAs: 31P NMR studies show the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus.

An enzyme preparation from Salmonella typhimurium catalyzes the conversion of 5-methylaminomethyl-2-thiouridine in tRNAs to 5-methylaminomethyl-2-selenouridine when supplemented with selenide and ATP. Similar preparations from a Salmonella mutant strain carrying a defective selD gene fail to catalyze this selenium substitution reaction. However, supplementation of the deficient enzyme preparation with the purified selD gene product (SELD protein) restored synthesis of seleno-tRNAs. In the absence of the complementary enzyme(s), the SELD protein catalyzes the synthesis of a labile selenium donor compound from selenide and ATP. 31P NMR studies show that among the products of this reaction are AMP and a compound containing selenium bonded to phosphorus. The reaction is completely dependent on the addition of both selenide and magnesium. The dependence of reaction velocity on ATP concentration shows sigmoidal kinetics, whereas dependence on selenide concentration obeys Michaelis-Menten kinetics indicating a Km value of 46 microM for selenide.

Adenosine Monophosphate↗
PubMed1992

Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli.

Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.

Amino Acid Oxidoreductases↗
PubMed1991

Glycine reductase protein C. Properties and characterization of its role in the reductive cleavage of Se-carboxymethyl-selenoprotein A.

The clostridial glycine reductase complex catalyzes the reductive deamination of glycine in an energy-conserving process that results in the esterification of orthophosphate. The complex consists of three protein components: selenoprotein A; protein B, a carbonyl group protein; and protein C, a sulfhydryl protein. The protein C component also catalyzes the arsenate-dependent decomposition of acetyl phosphate. Reaction of protein C with iodoacetate inhibits its ability to decompose acetyl phosphate, but this inactivation of the enzyme by alkylation is prevented in the presence of the substrate indicating the formation of an unreactive enzyme-bound acetylthiol ester. The Se-carboxy-methylselenocysteine residue of the selenoprotein A component of glycine reductase was generated by selective alkylation of the ionized selenol group at pH 6 with [14C]bromoacetate. Using this pure alkylated selenoprotein A as substrate, it was shown that protein C catalyzes the conversion of the [14C]carboxymethyl group, in selenoether linkage to protein A, to [14C]acetate in the presence of arsenate, dithiothreitol, and Mg2+. A procedure using hydrophobic chromatographic matrices was developed for the large scale isolation of protein C, and a number of the properties of the enzyme were determined.

Alkylation↗
PubMed1991

Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium.

Formate dehydrogenase H of Escherichia coli contains selenocysteine as an integral amino acid. We have purified a mutant form of the enzyme in which cysteine replaces selenocysteine. To elucidate the essential catalytic role of selenocysteine, kinetic and physical properties of the mutant enzyme were compared with those of wild type. The mutant and wild-type enzymes displayed similar pH dependencies with respect to activity and stability, although the mutant enzyme profiles were slightly shifted to more alkaline pH. Both enzymes were inactivated by reaction with iodoacetamide; however, addition of the substrate, formate, was necessary to render the enzymes susceptible to alkylation. Alkylation-induced inactivation was highly dependent on pH, with each enzyme displaying an alkylation vs. pH profile suggestive of an essential selenol or thiol. Both forms of the enzyme use a ping-pong bi-bi kinetic mechanism. The mutant enzyme binds formate with greater affinity than does the wild-type enzyme, as shown by reduced values of Km and Kd. However, the mutant enzyme has a turnover number which is more than two orders of magnitude lower than that of the native selenium-containing enzyme. The lower turnover number results from a diminished reaction rate for the initial step of the overall reaction, as found in kinetic analyses that employed the alternative substrate deuterioformate. These results indicate that the selenium of formate dehydrogenase H is directly involved in formate oxidation. The observed differences in kinetic properties may help explain the evolutionary conservation of selenocysteine at the enzyme's active site.

Cloning, Molecular↗
PubMed1991

Selenocysteyl-tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena.

Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.

Animals↗
PubMed1991

Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.

Amino Acid Oxidoreductases↗
PubMed1991

Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product.

The formate dehydrogenase (FDHF) of Escherichia coli is a selenocysteine-containing protein that occurs as a component of the formate-hydrogen lyase complex. The gene encoding this 80 kd polypeptide contains a TGA codon in the open reading frame. Several indirect lines of evidence showed earlier that the selenocysteine residue in the protein is inserted co-translationally in a TGA (UGA) dependent process. Direct proof that the selenocysteine is present in the polypeptide in the position corresponding to TGA as predicted from the gene sequence was obtained by automated amino acid sequence analysis of a 75Se-containing peptide isolated from the protein. Construction of a fusion gene comprising a small segment of the fdhF gene linked to the lacZ gene as reporter greatly facilitated isolation of the selenocysteine-containing protein. Subsequent cleavage of this isolated gene product with endoproteinase Asp-N gave rise to an easily purified small selenocysteine-containing peptide that was amenable to amino acid sequence analysis.

Amino Acid Sequence↗
PubMed1990

Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component.

The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence of exogenous electron acceptors. The complex consists of two separable enzymatic activities: a formate dehydrogenase and a hydrogenase. The formate dehydrogenase component (FDHH) of the formate-hydrogen lyase complex was purified to near homogeneity in two column chromatographic steps. The purified enzyme was composed of a single polypeptide of molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Metal analysis showed each mole of enzyme contained 3.3 g atoms of iron. Denaturation of FDHH released a compound which, when oxidized, displayed a fluorescence spectrum similar to that of the molybdopterin cofactor found in certain other enzymes. The enzyme contained selenium in the form of selenocysteine as determined by radioactive labeling of the enzyme with 75Se and amino acid analysis. FDHH activity was maximal between pH 7.5 and 8.5; however, the enzyme was maximally stable at pH 5.3-6.4 and highly unstable above pH 7.5. Nitrate and nitrite salts caused a drastic reduction in activity. Although azide inhibited FDHH activity, it also protected the enzyme from inactivation by oxygen.

Amino Acid Sequence↗
PubMed1990

In vitro incorporation of selenium into tRNAs of Salmonella typhimurium.

Broken-cell preparations of Salmonella typhimurium rapidly incorporated 75Se from 75SeO3(2-) into tRNA by an ATP-dependent process. Selenium incorporation in the presence of 50 microM 75SeO3(2-) (0.8-1 pmol per A260 unit) was enhanced by the selenocysteine precursor, O-acetyl-L-serine (to 3.7 pmol per A260 unit). This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of 75Se from 75SeO3(2-) was decreased by adding L-selenocysteine but not by adding the D isomer. When homologous bulk tRNA was added to the broken-cell preparations, an increased rate of 75Se labeling was observed. The supernatant fraction of the broken-cell preparation contained all of the enzymes required for this process. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that coeluted with authentic 5-methylaminomethyl-2-selenouridine.

Adenosine Triphosphate↗
PubMed1990

Biosynthesis of selenium-modified tRNAs in Methanococcus vannielii.

Selenium-containing nucleosides are natural components of several tRNA species in Methanococcus vannielii. In the present study, the incorporation of selenium from 75SeO3(2-) into these macromolecules was investigated in sonic extracts of M. vannielii. Nucleoside analysis of the 75Se-labeled tRNAs from these in vitro reaction mixtures demonstrated that the selenium was present in 75Se-labeled nucleosides identical to the two naturally occurring 2-selenouridines produced in vivo. Incorporation of selenium into these nucleosides was ATP-dependent and was maximal after 20 min. Addition of O-acetylserine enhanced the activity 2- to 3-fold, implicating a role for selenocysteine in the reaction. Added L-selenocysteine could function as a selenium donor, but the D isomer and DL-selenomethionine were inactive. RPC-5 chromatography of bulk tRNA isolated from M. vannielii grown on 75SeO3(2-) separated five major species of seleno-tRNAs. The amino acid-accepting activity of these tRNAs was investigated.

Amino Acids↗
PubMed1989

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%).

Animals↗
PubMed1989

Occurrence in vivo of selenocysteyl-tRNA(SERUCA) in Escherichia coli. Effect of sel mutations.

The selC gene of Escherichia coli codes for a novel tRNA species which is aminoacylated by L-serine and is required for the insertion of selenocysteine into proteins (Leinfelder, W., Zehelein, E., Mandrand-Berthelot, M.-A., and Böck, A. (1988) Nature 331, 723-725). As a first step toward the elucidation of the postulated pathway for selenocysteine formation from an L-serine residue esterified to tRNA, we have examined whether an increase in the selC gene dosage allows the demonstration of selenocysteyl-tRNA formation in vivo. To this end, cells of an E. coli strain carrying selC on a multicopy plasmid were labeled with [75Se]selenite, their tRNA was isolated and deacylated, and the hydrolysate was analyzed by thin layer chromatography and ion exchange chromatography. Both methods unequivocally demonstrated that the increase in the selC gene product concentration correlated with an augmented level of selenocysteine bound to tRNA. The formation of selenocysteine depended on the presence of functional products of the selA and selD genes but not of the selB gene. The selB gene product, therefore, may have a function in the decoding step itself.

Base Sequence↗