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J R Andreesen

Publications and source records attributed to J R Andreesen.

At least 55 records · Page 3Linked to original sources
PubMed1992

Utilization of organic acids and amino acids by species of the genus Bacillus: a useful means in taxonomy.

The type strains of 26 Bacillus species were investigated with respect to their capability to utilize 48 organic acids and 20 amino acids by using phenol red indicator plates and an amino acid analyzer, respectively. In addition, strains of Bacillus sphaericus were found to utilize many organic acids and amino acids in contrast to carbohydrates which are routinely checked. The results obtained indicated an individual utilization pattern for every species investigated, pointing to a useful taxonomic application of these tests for the genus Bacillus.

Amino Acids↗
PubMed1991

Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria.

Eighteen different aerobic bacteria were isolated which utilized quinoline as sole source of carbon, nitrogen, and energy. Attempts were unsuccessful at isolating anaerobic quinoline-degrading bacteria. The optimal concentration of quinoline for growth was in the range of 2.5 to 5 mM. Some organisms excreted 2-hydroxyquinoline as the first intermediate. Hydroxylation of quinoline was catalyzed by a dehydrogenase which was induced in the presence of quinoline or 2-hydroxyquinoline. Quinoline dehydrogenase activity was dependent on the availability of molybdate in the growth medium. Growth on quinoline was inhibited by tungstate, an antagonist of molybdate. Partially purified quinoline dehydrogenase from Pseudomonas putida Chin IK indicated the presence of flavin, iron-sulfur centers, and molybdenum-binding pterin. Mr of quinoline dehydrogenase was about 300 kDa in all isolates investigated.

Bacteria, Aerobic↗
PubMed1991

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected].

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophilum and Clostridium litorale [corrected] was monitored by a new spectrophotometric assay. The procedure depended on a specific two- to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E. acidaminophilum by 75Se labeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.

Amino Acid Sequence↗
PubMed1991

Thioredoxin elicits a new dihydrolipoamide dehydrogenase activity by interaction with the electron-transferring flavoprotein in Clostridium litoralis and Eubacterium acidaminophilum.

The glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing dihydrolipoamide. The first one was found to be a genuine dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by dihydrolipoamide was the strongly preferred reaction. A second dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088-2095, 1990) and a thioredoxin. This enzyme system was responsible for most of the dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.

Amino Acid Sequence↗
PubMed1990

Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes.

Three different dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested.

Clostridium↗
PubMed1990

Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases.

Chromatography↗
PubMed1990

Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria.

Three electron-transferring flavoproteins were purified to homogeneity from anaerobic, amino acid-utilizing bacteria (bacterium W6, Clostridium sporogenes, and Clostridium sticklandii), characterized, and compared with the dihydrolipoamide dehydrogenase of Eubacterium acidaminophilum. All the proteins were found to be dimers consisting of two identical subunits with a subunit Mr of about 35,000 and to contain about 1 mol of flavin adenine dinucleotide per subunit. Spectra of the oxidized proteins exhibited characteristic absorption of flavoproteins, and the reduced proteins showed an A580 indicating a neutral semiquinone. Many artificial electron acceptors, including methyl viologen, could be used with NADPH as the electron donor but not with NADH. Unlike the enzyme of E. acidaminophilum, which exhibited by itself a dihydrolipoamide dehydrogenase activity (W. Freudenberg, D. Dietrichs, H. Lebertz, and J. R. Andreesen, J. Bacteriol. 171:1346-1354, 1989), the electron-transferring flavoprotein purified from bacterium W6 reacted with lipoamide only under certain assay conditions, whereas the proteins of C. sporogenes and C. sticklandii exhibited no dihydrolipoamide dehydrogenase activity. The three homogeneous electron-transferring flavoproteins were very similar in their structural and biochemical properties to the dihydrolipoamide dehydrogenase of E. acidaminophilum and exhibited cross-reaction with antibodies raised against the latter enzyme. N-terminal sequence analysis demonstrated a high degree of homology between the dihydrolipoamide dehydrogenase of E. acidaminophilum and the electron-transferring flavoprotein of C. sporogenes to the thioredoxin reductase of Escherichia coli. Unlike these proteins, the dihydrolipoamide dehydrogenases purified from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and C. sporogenes exhibited a high homology to dihydrolipoamide dehydrogenases known from other organisms.

Amino Acid Sequence↗
PubMed1989

Molybdenum Involvement in Aerobic Degradation of 2-Furoic Acid by Pseudomonas putida Fu1.

An organism identified as Pseudomonas putida was isolated from an enrichment culture with 2-furoic acid as its sole source of carbon and energy. The organism contained a 2-furoyl-coenzyme A (CoA) synthetase to form 2-furoyl-CoA and a 2-furoyl-CoA dehydrogenase to form 5-hydroxy-2-furoyl-CoA as the first two enzymes involved in the degradation. Tungstate, the specific antagonist of molybdate, decreased growth rate and consumption of 2-furoic acid but had no influence on growth with succinate. Correspondingly, the 2-furoyl-CoA dehydrogenase activity decreased when the organism was grown on 2-furoic acid in the presence of increasing amounts of tungstate. The addition of molybdate reversed the negative effect on 2-furoyl-CoA dehydrogenase activity, which points to the involvement of a molybdoenzyme in this reaction. Both enzymes studied were inducible. No plasmid was detected in this organism.

Journal Article↗
PubMed1989

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum.

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-14C]riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band. NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase. This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations. The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity. Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.

Amino Acid Oxidoreductases↗
PubMed1989

Purification and partial characterization of the glycine decarboxylase multienzyme complex from Eubacterium acidaminophilum.

The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum. By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an alpha 2 beta 2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively. In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity. All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction. The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate. The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2. Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2. Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity. Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.

Amino Acid Oxidoreductases↗
PubMed1984

Comparative studies on physiology and taxonomy of obligately purinolytic clostridia.

Eleven strains of obligately purinolytic clostridia have been studied with respect to their assignment to the three type strains of Clostridium acidiurici, C. cylindrosporum, and C. purinolyticum. DNA/DNA-hybridization proved to be the method of choice for differentiation whereas phenotypic characteristics such as spore morphology, substrate spectra, nutritional requirements, product formation, and sensitivity against various antibiotics did not allow unequivocal identification. All strains depended on selenite for growth.

Anti-Bacterial Agents↗
PubMed1983

Purine and glycine metabolism by purinolytic clostridia.

Cell extracts of Clostridium acidiurici, C. cylindrosporum, and C. purinolyticum converted purine, hypoxanthine, 2-hydroxypurine, 6,8-dihydroxypurine, and uric acid into xanthine by the shortest possible route. Adenine was transformed to xanthine only by C. purinolyticum, whereas the other two species formed 6-amino-8-hydroxypurine, which was neither deaminated nor hydroxylated further. 8-Hydroxypurine was formed from purine by all three species. Xanthine dehydrogenase activity was constitutively expressed by C. purinolyticum. Due to the lability of the enzyme activity, comparative studies could not be done with a purified preparation. All enzymes reported to be involved in formiminoglycine metabolism of C. acidiurici and C. cylindrosporum were present in C. purinolyticum. However, glycine was reduced directly to acetate in all three species, as indicated by radiochemical data and by the detection of glycine reductase in cell extracts of C. cylindrosporum and C. purinolyticum. The expression of glycine reductase and the high ratio of glycine fermented to uric acid present points to an energetic advantage for the glycine reductase system, which is expressed when selenium compounds are added to the growth media.

Clostridium↗
PubMed1982

Anaerobic degradation of uric acid via pyrimidine derivatives by selenium-starved cells of Clostridium purinolyticum.

Clostridium purinolyticum decomposed uric acid via pyrimidine derivatives under selenium starvation conditions. Products were acetate, formate, glycine, ammonia, and CO2. 4,5-Diaminouracil could be identified as an intermediate after converting the labile substance into 6,7-dimethyllumazine. The breakdown of uric acid was inhibited by EDTA. High-pressure liquid chromatography methods have been developed for the simultaneous determination of uric acid, 4,5-diaminouracil, and 6,7-dimethyllumazine. The significance of the new pathway is discussed.

Anaerobiosis↗
PubMed1982

Selenium-dependent growth and glycine fermentation by Clostridium purinolyticum.

Clostridium purinolyticum fermented glycine as a sole carbon and energy source according to the equation: 4 Glycine + 2H2O leads to 3 Acetate + 2CO2 + 4NH3. The organism required adenine as a supplement and selenium compounds as micronutrients for growth. The molar growth yield on glycine was 6.5 g dry wt. Radiochemical and enzymic investigations revealed a new fermentation pathway for glycine in which 1 mol glycine was completely oxidized to CO2 and the generated reducing equivalents were used to reduce a further 3 mol glycine to acetate via the glycine reductase system. This reaction was associated with the formation of ATP.

Amino Acid Oxidoreductases↗
PubMed1979

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum.

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mumol/min x mg of protein. Cells grown on uric acid in the presence of 10(-7) M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10(-5) M). The supplementation with 10(-7) M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10(-5) M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium. In C. cyclindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mumol/min x mg of protein. The addition of 10(-7) M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10(-7) M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.

Cell-Free System↗
PubMed1978

Fumarate reductase of Clostridium formicoaceticum. A peripheral membrane protein.

When Clostridium formicoaceticum was grown on fumarate or L-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2-3.5 U per mg of protein. Reduced benzyl viologen, FMNH2 and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates. The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. There results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.

Acetate Kinase↗
PubMed1978

Fermentation of fumarate and L-malate by Clostridium formicoaceticum.

The fermentation of fumarate and L-malate by Clostridium formicoaceticum was investigated. Growing and nongrowing cells degraded fumarate by dismutation to succinate, acetate, and CO2; on the other hand, only small amounts of succinate were detected when the organism was grown on L-malate. This dicarboxylic acid was mainly converted to acetate and CO2. The fermentation balances were modified if bicarbonate or formate were present in the medium. When C. formicoaceticum was grown in the presence of both dicarboxylic acids, fumarate was consumed before L-malate. The latter was mainly converted to acetate, whereas fumarate was fermented to acetate and succinate. Molar growth yields were determined to be 6 g of dry weight per mol of fumarate and 8 g of dry weight per mol of L-malate fermented.

Acetates↗