Purification of carbon monoxide dehydrogenase, a nickel enzyme from Clostridium thermocaceticum

Carbon monoxide dehydrogenase (CO dehydrogenase) has been purified from the homoacetate-fermenting bacterium, Clostridium thermoaceticum. By use of 63Ni, it has been determined that the dehydrogenase is a metallo nickel enzyme. Nickel was rapidly taken up by the organism and most of the ingested metal was found to be incorporated into CO dehydrogenase. As estimated by gel filtration, the native enzyme has a molecular weight of 410,000. Ferredoxin and a membrane-bound b-type cytochrome, both obtained from C. thermoaceticum, are rapidly reduced by the enzyme in the presence of carbon monoxide and both are considered to be native electron carriers. FMN and Desulfovibrio vulgaris cytochrome c3 were also reduced by the enzyme, while spinach ferredoxin, FAD, NAD, and NADP were not. CO dehydrogenase activity was not appreciably affected by propyl iodide, methyl iodide, carbon tetrachloride, or metal chelators, but was reversibly inhibited by KCN. A method for the in situ assay of CO dehydrogenase in polyacrylamide gels is presented.     

Levels of enzymes involved in the synthesis of acetate from CO2 in Clostridium thermoautotrophicum

The acetogenic bacterium Clostridium thermoautotrophicum, grown on methanol, glucose, or CO2-H2, contained high levels of corrinoids, formate dehydrogenase, tetrahydrofolate enzymes, carbon monoxide dehydrogenase, and hydrogenase. Cell-free extracts catalyzed pyruvate-dependent formation of acetate from methyltetrahydrofolate. These results suggest that C. thermoautotrophicum synthesizes acetate from CO2 via a formate-tetrahydrofolate-corrinoid pathway.     

Purification and characterization of carbon monoxide dehydrogenase, a nickel, zinc, iron-sulfur protein, from Rhodospirillum rubrum

Carbon monoxide dehydrogenase (CO dehydrogenase) from Rhodospirillum rubrum was shown to be an oxygen-sensitive, nickel, iron-sulfur, and zinc-containing protein that was induced by carbon monoxide (CO). The enzyme was purified 212-fold by heat treatment, ion-exchange, and hydroxylapatite chromatography and preparative gel electrophoresis. The purified protein, active as a monomer of Mr = 61,800, existed in two forms that were comprised of identical polypeptides and differed in metal content. Form 1 comprised 90% of the final activity, had a specific activity of 1,079 mumol CO oxidized per min-1 mg-1, and contained 7 iron, 6 sulfur, 0.6 nickel, and 0.4 zinc/monomer. Form 2 had a lower specific activity (694 mumol CO min-1 mg-1) and contained 9 iron, 8 sulfur, 1.4 nickel, and 0.8 zinc/monomer. Reduction of either form by CO or dithionite resulted in identical, rhombic ESR spectra with g-values of 2.042, 1.939, and 1.888. Form 2 exhibited a 2-fold higher integrated spin concentration, supporting the conclusion that it contained an additional reducible metal center(s). Cells grown in the presence of 63NiCl2 incorporated 63Ni into CO dehydrogenase. Although nickel was clearly present in the protein, it was not ESR-active under any conditions tested. R. rubrum CO dehydrogenase was antigenically distinct from the CO dehydrogenases from Methanosarcina barkeri and Clostridium thermoaceticum.     

Carbon monoxide-driven electron transport in Clostridium thermoautotrophicum membranes.

Membrane vesicles of Clostridium thermoautotrophicum prepared by osmotic lysis after lysozyme treatment contained carbon monoxide dehydrogenase and methylenetetrahydrofolate dehydrogenase with specific activities three- to fourfold higher than the specific activity of the cytoplasm. The membrane-associated carbon monoxide dehydrogenase mediated the reduction with CO or the oxidation with CO2 of b-type cytochromes and other electron carriers in the membrane.     

Properties of purified carbon monoxide dehydrogenase from Clostridium thermoaceticum, a nickel, iron-sulfur protein

Carbon monoxide dehydrogenase from Clostridium thermoaceticum has been purified to homogeneity using a strict anaerobic procedure. The enzyme has a molecular weight of about 440,000 and it consists of three each of two different subunits giving the composition alpha 3 beta 3. The molecular weight of the alpha-subunit is 78,000 and that of the beta-subunit is 71,000. Pore limit gel electrophoresis gave a molecular weight of 161,000 indicating that the enzyme dissociates to form a dimer with an alpha beta structure. The dimer apparently contains per mol 2 nickel, 1 zinc, 11 iron, and 14 acid-labile sulfur. The anaerobic enzyme has an iron-sulfur type spectrum, which is changed in the presence of the substrate, CO. In the presence of oxygen, which destroys the activity or CO2, the spectrum is that of a typical iron-sulfur protein. Under acidic conditions a low molecular weight nickel factor separates from the enzyme. Viologens, methylene blue, ferredoxin, flavodoxin, and rubredoxin serve as electron acceptors. Of these rubredoxin is by far the most efficient. The enzyme has a pH optimum around 8.4. At this pH and 50 degrees C under 100% CO atmosphere, the apparent Km for methyl viologen is 3.03 mM and Vmax is 750 mumols of CO oxidized min-1 mg-1. Cyanide and methyl iodide inhibit the enzyme. CO reverses the cyanide inhibition but promotes the reaction with methyl iodide. The pure enzyme has no hydrogenase or formate dehydrogenase activity.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

Isolation of carbon monoxide dehydrogenase from Acetobacterium woodii and comparison of its properties with those of the Clostridium thermoaceticum enzyme.

An oxygen-labile carbon monoxide dehydrogenase was purified to at least 98% homogeneity from fructose-grown cells of Acetobacterium woodii. Gel filtration and electrophoresis experiments gave molecular weights of 480,000 and 153,000, respectively, of the active enzyme. The molecular weights for the subunits are 80,000 and 68,000; the subunits occur in equal proportion. The small subunit of the A. woodii enzyme differs in size from that of the Clostridium thermoaceticum enzyme; however, the large subunits are similar. The specific activity of the A. woodii enzyme, measured at 30 degrees C and pH 7.6, is 500 mumol of CO oxidized min-1 mg-1 with 20 mM methyl viologen as the electron acceptor. Analysis revealed (number per dimer) iron (9), acid-labile sulfide (12), nickel (1.4), and magnesium or zinc (1). This metal content is quite similar to that of the C. thermoaceticum enzyme (Ragsdale et al., J. Biol. Chem. 258:2364-2369, 1983). The nickel as well as the iron-sulfur clusters are redox-active, as was found for the C. thermoaceticum enzyme (Ragsdale et al., Biochem. Biophys. Res. Commun. 108:658-663, 1982). CO can reduce and CO2 can oxidize the iron-sulfur clusters. The enzyme is inhibited by cyanide, but CO2 in the presence of reduced methyl viologen or CO alone can reverse or prevent this inhibition. Several ferredoxins, flavodoxin, and rubredoxin and some artificial electron carriers were tested for their relative rates of reaction with the CO dehydrogenases from A. woodii, C. thermoaceticum, and Clostridium formicoaceticum. Rubredoxin was by far the most reactive acceptor and is proposed to be the primary natural electron carrier for the acetogenic CO dehydrogenases.     

Clostridium beijerinckii and Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87-93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.     

Butanol-Ethanol Dehydrogenase and Butanol-Ethanol-Isopropanol Dehydrogenase: Different Alcohol Dehydrogenases in Two Strains of Clostridium beijerinckii (Clostridium butylicum)

Alcohol-producing strains of Clostridium beijerinckii (Clostridium butylicum) produce, besides acetone, either n-butanol and ethanol or n-butanol, ethanol, and isopropanol as their characteristic products. Alcohol dehydrogenase has been isolated from a strain (NRRL B593) of C. beijerinckii producing isopropanol and from a strain (NRRL B592) not producing isopropanol. Butanol-ethanol dehydrogenase activities were present in both strains, but isopropanol dehydrogenase activity was present only in the isopropanol-producing strain. The butanol-ethanol dehydrogenase of strain NRRL B592 had M(r) 66,000 and a K(m) of 6 muM for butyraldehyde. In contrast, the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593 had a M(r) 100,000 and K(m)s of 9.5 and 1.0 mM for butyraldehyde and acetone, respectively. In a purification by four different types of separatory methods (DEAE-cellulose, hydroxyapatite, Sephacryl S-300, and Matrex Gel Red A), butanol-ethanol-isopropanol dehydrogenase activities of strain NRRL B593 were purified up to 200-fold (10 to 30% yield), and these activities were not separated. Gel electrophoresis followed by activity stain also revealed distinct mobilities for the butanol-ethanol dehydrogenase of strain NRRL B592 and the butanol-ethanol-isopropanol dehydrogenase of strain NRRL B593. In cell extracts from both strains, a higher alcohol dehydrogenase activity was measured with NADP(H) than with NAD(H). The 150- to 200-fold-purified alcohol dehydrogenase from strain NRRL B593 did not show any NAD(H)-linked activities. The K(m) for NADPH was 31 muM (with butyraldehyde as cosubstrate) and 18 muM (with acetone as cosubstrate) for the alcohol dehydrogenase of strain NRRL B593. This study showed that the alcohol dehydrogenases from two strains of C. beijerinckii differed significantly.     

Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum

Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C. innocuum had a pH optimum of 5.0. The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of 17 nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate. delta 4-3-Ketosteroid-5 beta-reductase from C. innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives. A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography. 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C. innocuum was oxygen sensitive and required NADH for activity. An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase. However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique.     

Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.     

Arginine and ornithine catabolism by Clostridium botulinum

Clostridium botulinum 62-A was shown to catabolize l-arginine via citrulline to ornithine, NH(3), and CO(2). The individual enzymes of the dihydrolase system were all demonstrated in extracts of cells, spores, and germinated spores. There was no liberation of urea from l-arginine, so no functional arginase enzyme is present, but there was some transamidinase activity in cell extracts. l-Ornithine was degraded at a significant rate by cells grown in an l-ornithine-supplemented medium; it was partially decarboxylated to putrescine and partially fermented to NH(3), CO(2), volatile acids, and delta-aminovaleric acid. Results from the fermentation of l-ornithine-C(14), -1-C(14), and -2-C(14) demonstrated that essentially all of the CO(2) was derived from carbon 1, and volatile acids from carbons 2 to 5. Assays for the products of l-ornithine-C(14) fermentation revealed that the volatile acids consisted of acetate, propionate, valerate, and butyrate (in order of decreasing concentrations), and that delta-amino-valerate was the primary reduced product. A small amount of citrulline was formed during the fermentation. The carbon and redox balances indicated that l-ornithine is fermented as a single substrate. Preliminary experiments demonstrated that the fermentation of l-ornithine is carried out by cell extracts with the production of volatile acids.     

Clostridium beijerinckii and Clostridium difficile Detoxify Methylglyoxal by a Novel Mechanism Involving Glycerol Dehydrogenase

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.     

Precipitation of cadmium by Clostridium thermoaceticum.

Cadmium at an initial concentration of 1 mM was completely precipitated by cultures of Clostridium thermoaceticum in complex medium. The precipitation was energy dependent and required cysteine, although cysteine alone did not act as a growth substrate. Electron microscopic analysis revealed localized areas of precipitation at the surfaces of nonstarved cells as well as precipitate in the surrounding medium. The addition of cadmium had no apparent effect on growth or acetogenesis. However, nickel and cadmium were synergistically toxic at a concentration (1 mM) at which neither alone was toxic. The amount of protein extracted from cadmium-treated cultures was twofold higher than that in control extracts, and the amount of total sulfide was fourfold higher in cultures containing cadmium than in control cultures. Comparable levels of cysteine desulfhydrase activity were observed in extracts of both cadmium-treated and control cultures, but the enzyme activity was expressed maximally about 24 h earlier in the cadmium-treated cultures than in the untreated controls.     

Fermentation of Fructose and Synthesis of Acetate from Carbon Dioxide by Clostridium formicoaceticum

Clostridium formicoaceticum ferments fructose labeled with (14)C in carbon 1, 4, 5, or 6 via the Embden Meyerhof pathway. In fermentations of fructose in the presence of (14)CO(2), acetate is formed labeled equally in both carbons. Extracts convert the methyl groups of 5-methyltetrahydrofolate and methyl-B(12) to the methyl group of acetate in the presence of pyruvate. Formate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and 5,10-methylenetetrahydrofolate reductase are present in extracts of C. formicoaceticum. These enzymes are needed for the conversion of CO(2) to 5-methyltetrahydrofolate. It is proposed that acetate is totally synthesized from CO(2) via the reactions catalyzed by the enzymes listed above and that 5-methyltetra-hydrofolate and a methylcorrinoid are intermediates in this synthesis.     

Utilization of Glucose by Clostridium thermocellum: Presence of Glucokinase and Other Glycolytic Enzymes in Cell Extracts

Clostridium thermocellum was shown to ferment glucose in a medium containing salts and 0.5% yeast extract. An active glucokinase was obtained with improved conditions for growth, assay, and preparation of cell extracts. Cell extracts appear to contain a glucokinase inhibitor that interferes with the assays at high protein concentrations. Glucokinase activity is stimulated about 60% by pretreatment with dithiothreitol. Little or no fructokinase or mannokinase activity was detected in cell extracts. The absence of glucokinase in mannitol-grown cells, the increase in glucokinase activity upon incubation of cell suspensions with glucose, and the lack of increase in activity when chloramphenical is added are evidence that glucokinase is an inducible enzyme. The following enzymes were detected in cell extracts (the enzyme activities are shown in parentheses are micromoles per minute per milligram or protein at 27 C): glucokinase (0.48), phosphoglucose isomerase (0.73), fructose 6-phosphate kinase (0.24), fructose diphosphate aldolase (0.59), glyceraldehyde 3-phosphate dehydrogenase (0.53), triose phosphate isomerase (0.13), phosphoglycerate kinase (0.20), phosphoglycerate mutase (0.20), enolase (0.28), pyruvic kinase (0.13), and lactic dehydrogenase (0.13). Glucose 6-phosphate dehydrogenase activity was absent or very low (0.0002) and 6-phosphogluconate dehydrogenase activity also was relatively low (0.015). From these data, it is proposed that carbohydrate metabolism in C. thermocellum proceeds by the Embden-Meyerhof pathway.     

Formate dehydrogenase from Clostridium acidiurici

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO(2) was not accomplished.     

Glucose Degradation in Clostridium perfringens Type A

Cell-free extracts of Clostridium perfringens were found to contain all the enzymes of the Embden-Meyerhof pathway of glycolysis in addition to lactic acid dehydrogenase and the pyruvate-clastic system. Neither glucose-6-phosphate dehydrogenase nor 6-phosphogluconate dehydrogenase activities could be demonstrated, suggesting the absence of the conventional hexose-monophosphate pathway in this organism.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.