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T C Stadtman

Publications and source records attributed to T C Stadtman.

At least 91 records · Page 5Linked to original sources
PubMed1987

Purification and properties of carbon monoxide dehydrogenase from Methanococcus vannielii.

Carbon monoxide dehydrogenase was purified to homogeneity from Methanococcus vannielii grown with formate as the sole carbon source. The enzyme is composed of subunits with molecular weights of 89,000 and 21,000 in an alpha 2 beta 2 oligomeric structure. The native molecular weight of carbon monoxide dehydrogenase, determined by gel electrophoresis, is 220,000. The enzyme from M. vannielii contains 2 g-atoms of nickel per mol of enzyme. Except for its relatively high pH optimum of 10.5 and its slightly greater net positive charge, the enzyme from M. vannielii closely resembles carbon monoxide dehydrogenase isolated previously from acetate-grown Methanosarcina barkeri. Carbon monoxide dehydrogenase from M. vannielii constitutes 0.2% of the soluble protein of the cell. By comparison the enzyme comprises 5% of the soluble protein in acetate-grown cells of M. barkeri and approximately 1% in methanol-grown cells.

Aldehyde Oxidoreductases↗
PubMed1986

Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA.

A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli. The synthesis of mnm5se2U is optimum (4 mol/100 mol tRNA) when selenium is present at about 1 microM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 microM) levels of selenium. Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E. coli cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions. The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position. In E. coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated. Broken cell preparations of E. coli synthesize mnm5se2U in tRNAs by an ATP-dependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium.

Chromatography, High Pressure Liquid↗
PubMed1986

Diol metabolism and diol dehydratase in Clostridium glycolicum.

Levels of the five enzymes involved in the fermentation of 1,2-ethanediol and 1,2-propanediol in the strictly anaerobic bacterium, Clostridium glycolicum, were investigated. All enzymes with the exception of the first enzyme in the pathway, diol dehydratase, were found to be constitutive, stable to exposure to oxygen, and present in the cytosol. Diol dehydratase was found to be extremely oxygen sensitive and strongly associated with the cell membrane. Treatment with ionic and nonionic detergents, butanol, phospholipase A2, or osmotic shock procedures failed to solubilize any diol dehydratase activity. Limited proteolysis using subtilisin released small amounts of activity. Diol dehydratase was found to be specific for 1,2-ethanediol and 1,2-propanediol and required the addition of a reducing agent for maximal activity. The enzyme was strongly inhibited by low concentrations of EDTA, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, o-phenanthroline, hydroxylamine, hydroxyurea, and sulfhydryl reagents. Addition of adenosylcobalamin or high levels of intrinsic factor did not affect the reaction rate. Irradiation with light also did not inhibit the enzyme activity. These results suggest that the catalytic mechanism of diol dehydratase from C. glycolicum does not involve a cobamide coenzyme.

Acetate Kinase↗
PubMed1986

Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli.

The gene (fdhF) coding for the selenopolypeptide of the benzylviologen-linked formate dehydrogenase of Escherichia coli was cloned and its nucleotide sequence was determined. The fdhF gene contains, within an open reading frame coding for a protein of 715 amino acids (calculated molecular weight, 79,087), an opal (UGA) nonsense codon in amino acid position 140. Existence of this nonsense codon was confirmed by physical recloning and resequencing. Internal and terminal deletion clones and lacZ fusions of different N-terminal parts of fdhF were constructed and analyzed for selenium incorporation. Selenylated truncated polypeptide chains or beta-galactosidase fusion proteins were synthesized when the deletion clones or gene fusions, respectively, contained the fdhF gene fragment coding for the selenopolypeptide sequence from amino acid residue 129 to amino acid residue 268. Translation of the lacZ part of the fusions required the presence of selenium in the medium when the N-terminal fdhF part contained the UGA codon and was independent of the presence of selenium when a more upstream part of fdhF was fused to lacZ. The results are consistent with a co-translational selenocysteine incorporation mechanism.

Aldehyde Oxidoreductases↗
PubMed1985

Incorporation and distribution of selenium into thiolase from Clostridium kluyveri.

Clostridium kluyveri incorporates selenium as selenomethionine into its acetoacetyl-CoA thiolase when grown in media containing normal sulfur-to-selenium ratios. Antibodies raised against the purified enzyme permitted quantitative immunoprecipitation of thiolase from crude cell extracts and thus facilitated the systematic analysis of the effects of wide variation in sulfur-to-selenium ratios on selenium incorporation into the enzyme. The extent of incorporation of selenium into thiolase was found to be dependent on the form of selenium supplied. When [75Se]selenomethionine was the source of selenium, the incorporation of selenium into thiolase was inversely proportional to the level of added methionine. However, similar levels of methionine failed to decrease the incorporation of selenium from selenite. To study the location of selenomethionine and methionine residues in the polypeptide chain of the enzyme, thiolase was prepared from cells cultured in the presence of H2 35SO4 or Na2 75SeO3. The 35S- or 75Se-labeled protein was treated with trypsin and the resulting peptides were isolated by reverse phase high performance liquid chromatography. The peptide maps of the enzyme indicated that selenium was distributed throughout the primary structure in a manner that paralleled methionine. From these studies, it is concluded that selenium occurs in thiolase adventitiously and is not required for any biological function.

Acetyl-CoA C-Acetyltransferase↗
PubMed1985

Purification and properties of a quinone-dependent p-nitrophenylphosphatase from Clostridium sticklandii.

A highly specialized phosphatase that depends on both a quinone (e.g., 2-methyl-1,4-napthoquinone) and a sulfhydryl compound for activity was purified to homogeneity from extracts of Clostridium sticklandii. Selective adsorption to Cibacron Blue-Sepharose 4B followed by elution with p-nitrophenylphosphate was an effective enrichment procedure. An affinity matrix containing vitamin K5 (4-amino-2-methyl-1-naphthol) covalently attached to Sepharose 4B selectively retained the enzyme and was also used in its purification. The only known substate for the enzyme, p-nitrophenylphosphate, is hydrolyzed to equivalent amounts of orthophosphate and p-nitrophenol. Although a protein phosphotyrosine residue seemed a likely candidate as the natural substrate, the enzyme failed to hydrolyze 32P-labeled phosphotyrosine residues in casein, in vinculin, or in denatured glutamine synthetase. Also, free O-phosphotyrosine and numerous phosphate esters that serve as substrates for common phosphomonoesterases were not hydrolyzed. The molecular weight of the native enzyme, estimated by Sephacryl-S-200 gel chromatography, is 27,600. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed a single component with a molecular weight of 28,600. From the amino acid composition, a minimum molecular weight of 28,000 was calculated.

4-Nitrophenylphosphatase↗
PubMed1985

A selenium-containing nucleoside at the first position of the anticodon in seleno-tRNAGlu from Clostridium sticklandii.

In previous studies, the single selenonucleoside component of a selenium-containing tRNAGlu isolated from Clostridium sticklandii has been shown to be 5-methyl-aminomethyl-2-selenouridine. Here, we show that this selenonucleoside is most likely located at the "wobble" position of the anticodon of the clostridial seleno-tRNAGlu. Nuclease T1 digestion of this seleno-tRNAGlu generated one major selenium-containing oligonucleotide (25 bases long). The selenium-containing residue within this oligonucleotide was located by sequence analysis of the oligonucleotide before and after removal of selenium by treatment with cyanogen bromide. The sequence of this oligonucleotide, A-A-C-C-G-C-C-C-U-U+-U-C-A+C-G-G-C-G-G-U-A-A-C-A-G, is homologous to that of the Escherichia coli tRNAGlu2 from residues 27 to 50, including the anticodon region and the variable loop, except that the E. coli tRNA has 5-methylaminomethyl-2-thiouridine instead of the selenonucleoside.

Anticodon↗
PubMed1985

Stereochemical studies of a selenium-containing hydrogenase from Methanococcus vannielii: determination of the absolute configuration of C-5 chirally labeled dihydro-8-hydroxy-5-deazaflavin cofactor.

Reduction of 7,8-didemethyl-8-hydroxy-[5-2H]-5-deazariboflavin by the selenium-containing hydrogenase from Methanococcus vannielii gave a C-5 chirally labeled 1,5-dihydro derivative. The absolute configuration of the chiral label was shown to be (R) by comparison of the chemically degraded product with authentic samples of known absolute configurations. Therefore, the steric course of the enzymic reactions involving the 8-hydroxy-5-deazaflavin cofactor can be defined as follows: (a) reduction occurs on the si face of the 5-deazaflavin molecule; (b) oxidation proceeds by the abstraction of the pro-S hydrogen at C-5 of the 1,5-dihydro-5-deazaflavin. Thus, the selenium-containing hydrogenase and 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii are si face specific.

Euryarchaeota↗
PubMed1984

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine.

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.

Escherichia coli↗
PubMed1984

Distribution of two selenonucleosides among the selenium-containing tRNAs from Methanococcus vannielii.

Naturally occurring seleno-tRNAs from Methanococcus vannielii account for 13-20% of the total tRNA population. Two different selenium-modified nucleosides were detected in these seleno-tRNAs. Of the total selenium incorporated, about 60% is present in 5-methylaminomethyl-2-selenouridine, and the other 40% occurs in a second selenonucleoside with spectral characteristics typical of a derivative of 2-selenouridine. The 5-methylaminomethyl-2-selenouridine was found in the seleno-tRNA species present in the early fractions of a linear salt gradient elution profile from a reversed-phase chromatographic system 5 (RPC-5) column, whereas the second selenonucleoside occurred in the tRNA species eluted late in the profile.

Chromatography, High Pressure Liquid↗
PubMed1982

Analogues of 8-hydroxy-5-deazaflavin cofactor: relative activity as substrates for 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii.

The 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii was examined for its ability to catalyze the reduction of a number of 5-deazaflavin analogues of the natural cofactor. Comparison of the kinetic constants revealed certain substrate structure-reactivity relationships for the enzyme. The basic heterocyclic system of the natural cofactor 2,4-dioxopyrimido [4,5-b]quinoline was shown to be the minimum structural requirement since neither riboflavin nor 1,5-dideazariboflavin was reduced by the enzyme. The N-10 side chain of the natural cofactor was shown not to be essential since the enzyme could reduce 8-hydroxy-2,4-dioxopyrimido[4,5-b]quinoline. The study also indicated that there are some steric constraints at C-8 and C-7 with respect to interaction of the cofactor with the enzyme. Specifically, (a) the 8-methoxy derivative, in contrast to the 8-hydroxy compound, was not reduced and (b) the introduction of a substituent at C-7 resulted in a marked decrease in the rate of reduction. The importance of C-5 as the site for the electron entry was suggested by the finding that 5-methyl-deazariboflavin was not reduced. The latter inhibited the reduction of 5-deazariboflavin.

Euryarchaeota↗
PubMed1982

Isolation of a selenium-containing thiolase from Clostridium kluyveri: identification of the selenium moiety as selenomethionine.

Clostridium kluyveri grown in the presence of 1 muM Na2(75)SeO3 produces a thiolase that copurifies with 75Se. Based on several criteria, the selenium moiety in this protein is selenomethionine. The 75Se-labeled amino acid in acid hydrolysates of the radioactive protein cochromatographed with authentic selenomethionine on an amino acid analyzer and on TLC plates in acidic and basic solvents. Incubation with S-adenosylmethionine synthetase and ATP converted the 75Se-labeled amino acid to a radioactive basic product that was indistinguishable from authentic Se-adenosylselenomethionine by ion exchange and TLC. The native selenoenzyme, Mr 155,000-158,000, is composed of four subunits of Mr 38,000-40,000. Thiolase of similar molecular weight that is less acidic and lacks selenium is also produced by C. kluyveri. The factors that control the relative levels of the two enzymes in the cell have not been identified.

Amino Acids↗
PubMed1982

Selenium-containing tRNAGlu from Clostridium sticklandii: correlation of aminoacylation with selenium content.

Selenium-containing amino acid tRNAs are normal components of several bacterial tRNA populations. In Clostridium sticklandii seleno-nucleotides occur in at least four different tRNA species which account for 5--8% of the total tRNA population. One of these has been isolated in a highly purified form and shown to be an isoaccepting tRNAGlu. Experimental evidence indicates that the presence of the seleno-nucleotide in this tRNAGlu is essential for its acylation with glutamate.

Clostridium↗
PubMed1981

Selenium-dependent and selenium-independent formate dehydrogenases of Methanococcus vannielii. Separation of the two forms and characterization of the purified selenium-independent form.

Anaerobic oxidation of formate by Methanococcus vannielii is catalyzed by two readily separable formate dehydrogenases. One of these is a 105,000-dalton protein that contains molybdenum, iron, and acid-labile sulfide, but not selenium. The other is a high molecular weight complex composed of selenoporotein and molybdo-iron sulfur protein subunits. Selenium occurs in this selenoenzyme in the chemical form of selenocysteine residues. M. vannielii cells from selenium-deficient media contain the 105,000-dalton formate dehydrogenase. Marked stimulation of growth by selenite supplementation is correlated with the simultaneous appearance in the cells of the high molecular weight selenoprotein . enzyme complex. The latter is the predominant form in cells from media additionally supplemented with tungstate. Under these conditions partial replacement of molybdenum with tungsten appears to occur. Both formate dehydrogenases are maximally active at pH 8.5 to 9.2 and at 60 degrees C and are extremely oxygen-sensitive. They utilize as electron acceptors 8-hydroxy-5-deazaflavin, FMN, FAD, and viologen and tetrazolium dyes.

Aldehyde Oxidoreductases↗
PubMed1980

Stereochemical studies of 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii.

The purified 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii catalyzes an oxidation-reduction reaction between a novel 8-hydroxy-5-deazaflavin cofactor and nicotinamide adenine dinucleotide phosphate. The reaction was shown to be a direct hydride transfer process. Using stereospecifically 3H-labeled substrates, the steric course of this process was established to be S-specific with respect to the nicotinamide nucleotide. The 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii and the hydrogenase system in the cell-free extracts of Methanobacterium thermoautotrophicum recognize the same side, designated as A side, with respect to the prochiral center at C-5 of the dihydro-8-hydroxy-5-deazaflavin cofactor.

Euryarchaeota↗