The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase.

Purification of aldehyde oxidoreductase from C. thermoaceticum, the first detected enzyme able to reduce reversibly non-activated carboxylic acids to the corresponding aldehydes (White, H., Strobl, G., Feicht, R. & Simon, H. (1989) Eur. J. Biochem. 184, 89-96), results in the generation of multiple forms of the enzyme. The specific activities for the viologen-mediated dehydrogenation of butyraldehyde for the two main forms of the purification procedure are 530 and 450 U/mg. Two forms of the enzyme composed of alpha,beta- and alpha,beta,gamma-subunits, can be differentiated. The latter binds to red-Sepharose and can be eluted very specifically with NADPH. In contrast to the alpha,beta-types the trimeric forms also catalyse the reversible reduction of oxidised viologen with NADPH (VAPOR activity). The dimer alpha,beta can oligomerize and the alpha,beta,gamma-trimer can easily form various oligomers or split off the gamma-subunit. The apparent molecular masses of the subunits alpha,beta and gamma are 64, 14 and 43 kDa. The alpha,beta-form reveals an apparent molecular mass of 86 kDa containing about 29 iron, 25 acid-labile sulphur, 0.8 tungsten and forms about 1 mol pterine-6-carboxylic acid by permanganate oxidation. The corresponding values of the trimer showing a mass of 300 kDa, are about 82 Fe, 54 S, 3.4 W and 2.5 pterine-6-carboxylic acid. In addition, 1.7 mol of FAD could be found which seems to be a component of the gamma-subunit. The aldehyde oxidoreductase from C. thermoaceticum and that from C. formicoaceticum (White, H., Feicht, R., Huber, C., Lottspeich, F. & Simon, H. (1991) Biol. Chem. Hoppe-Seyler 372, 999-1005) show qualitative similarities as far as the Fe, S, W and pterin content and the broad substrate specificity are concerned. However, there are also surprisingly marked differences with respect to composition and amino-acid sequence.     

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.     

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.     

Molecular cloning and biochemical characterization of L-N-carbamoylase from Sinorhizobium meliloti CECT4114

An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents. The apparent Km values for N-carbamoyl-L-methionine and tryptophan were very similar (0.65 +/- 0.09 and 0.69 +/- 0.08 mM, respectively), although the rate constant was clearly higher for the L-methionine precursor (14.46 +/- 0.30 s(-1)) than the L-tryptophan one (0.15 +/- 0.01 s(-1)). The enzyme also hydrolyzed N-formyl-L-methionine (kcat/Km = 7.10 +/- 2.52 s(-1) x mM(-1)) and N-acetyl-L-methionine (kcat/Km = 12.16 +/- 1.93 s(-1) x mM(-1)), but the rate of hydrolysis was lower than for N-carbamoyl-L-methionine (kcat/Km = 21.09 +/- 2.85). This is the first L-N-carbamoylase involved in the 'hydantoinase process' that has hydrolyzed N-carbamoyl-L-cysteine, though less efficiently than N-carbamoyl-L-methionine. The enzyme did not hydrolyze ureidosuccinic acid or 3-ureidopropionic acid. The native form of the enzyme was a homodimer with a molecular mass of 90 kDa. The optimum conditions for the enzyme were 60 degrees C and pH 8.0. Enzyme activity required the presence of divalent metal ions such as Ni2+, Mn2+, Co2+ and Fe2+, and five amino acids putatively involved in the metal binding were found in the amino acid sequence.     

Cytochrome b from Escherichia coli nitrate reductase. Its properties and association with the enzyme complex

Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms. The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification. A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome. The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels. The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively. Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved. These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm. Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin. The renatured cytochrome yielded absorption spectra similar to the holoenzyme. The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate. It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.     

Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non-activated carboxylic acids to aldehydes

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non-activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 microM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum. Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red-Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity. The very labile enzyme has been enriched by a factor of about 145 by binding to octyl-Sepharose and further chromatographic separation by red-Sepharose and FPLC using Mono-Q and phenyl-Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.     

Assimilatory nitrate reductase from the haloarchaeon Haloferax mediterranei: purification and characterisation

Haloferax mediterranei can use nitrate as sole nitrogen source during aerobic growth. We report here the purification and biochemical characterisation of the assimilatory nitrate reductase (EC 1.6.6.2) from H. mediterranei. The enzyme, as isolated, was composed of two subunits (105+/-1.3 kDa and 50+/-1.3 kDa) and behaved as a dimer during gel filtration (132+/-6 kDa). A pH of 9 and elevated temperatures up to 80 degrees C (at 3.1 M NaCl) are necessary for optimum activity. The enzyme stability and activity of the enzyme depend upon the salt concentration. Reduced methyl viologen was as effective as the natural electron donor ferredoxin in the catalytic process. In contrast, NADPH and NADH, which are electron donors in nitrate reductases from different non-photosynthetic bacteria, were ineffective.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

Enoate reductases of Clostridia. Cloning, sequencing, and expression

The enr genes specifying enoate reductases of Clostridium tyrobutyricum and Clostridium thermoaceticum were cloned and sequenced. Sequence comparison shows that enoate reductases are similar to a family of flavoproteins comprising 2,4-dienoyl-coenzyme A reductase from Escherichia coli and old yellow enzyme from yeast. The C. thermoaceticum enr gene product was expressed in recombinant Escherichia coli cells growing under anaerobic conditions. The recombinant enzyme was purified and characterized.     

Dihydro-orotase from Clostridium oroticum. Purification and reversible removal of essential zinc.

A new purification procedure involving five column-chromatography steps is described for dihydro-orotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) from Clostridium oroticum (A.T.C.C. 25750). The native purified enzyme is a dimer of Mr 102 000 and contains 4.0 +/- 0.3 g-atoms of zinc/mol of dimer. These observations agree with those reported previously [Taylor, Taylor, Balch & Gilchrist (1976) J. Bacteriol. 127, 863-873]. It is conclusively demonstrated that dihydro-orotase is a zinc metalloenzyme. Zinc is reversibly removed by treatment with chelators in phosphate buffer at pH 6.5, as demonstrated by atomic absorption spectrophotometry and decrease of enzyme activity. The specific activity is linearly dependent on zinc content. Addition of ZnSO4 to the chelator-treated enzyme results in regain of the normal complement of zinc and enzyme activity. Kinetic properties of the reconstituted enzyme are indistinguishable from those of the native enzyme. The amino acid composition of the homogeneous enzyme suggests that the zinc atoms occupy different environments.     

On a new artificial mediator accepting NADP(H) oxidoreductase from Clostridium thermoaceticum

The purification and partial characterisation of an NADP(H) dependent artificial mediator accepting pyridine nucleotide oxidoreductase (AMAPOR) from the anaerobic Clostridium thermoaceticum is described. Depending on the redox potential of the artificial mediators the AMAPOR is able to regenerate NADP+ or NADPH rendering the enzyme useful for preparative work applying NADP(H) dependent oxidoreductases. At 37 degrees C crude extracts of C. thermoaceticum have an AMAPOR activity of 5-7 U mg(-1). This is 28 degrees under the optimal growth temperature of this microrganism. Out of apparently more than 10 AMAPOR active proteins in the crude cell extracts visible after electrophoresis and activity staining on the gel, two of these proteins were isolated. They seem to be two different oligomers. According to gel electrophoresis they show apparent molecular masses of about 200 and 400 kDa. These two forms showed after SDS gel electrophoresis two monomers with apparent molecular masses of 42 and 56 kDa which we call alpha and beta. The two oligomers may have the compositions alpha2beta2 and alpha4beta4. They contain Fe/S cluster and FAD. Various amounts of the FAD were lost during the purification procedure. This loss is partially reversible after addition of FAD. The AMAPOR reacts with rather different artificial mediators such as viologens, quinones e.g. 1,4-benzoquinone or anthraquinone-2,6-disulphonate, 2,6-dichloro-indophenol and clostridial rubredoxin. Two different ferredoxins from C. thermoaceticum, oxygen or lipoamide are no substrates indicating the here described AMAPOR is not a diaphorase in the usual sense.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Aldehyde oxidation in human placenta. Purification and properties of 1-pyrroline-5-carboxylate dehydrogenase.

The human placenta contains a considerable amount of 1-pyrroline-5-carboxylate dehydrogenase (23 +/- 6 micrograms/g; n = 12), about 25% of the concentration present in liver. The enzyme is the only form in placenta that oxidizes short- and medium-chain aldehydes, which facilitates its purification from this organ. It can be purified to homogeneity by successive chromatographies on DEAE-cellulose, 5'-AMP-Sepharose and Sephacryl S-300. From 500 g of tissue, about 2.1 units of enzyme can be obtained with a 12% yield. Placental 1-pyrroline-5-carboxylate dehydrogenase is a dimer of Mr-63,000 subunits. It exhibits a pI of 6.80-6.65, and is specific for 1-pyrroline-5-carboxylate, the cyclic form of glutamate gamma-semialdehyde (Km = 0.17 mM, kcat. = 870 min-1), although it also oxidizes short-chain aliphatic aldehydes such as propionaldehyde (Km = 24 mM, kcat. = 500 min-1). These properties are very close to those of the liver enzyme, indicating a strong similarity between the enzyme forms from both organs. The enzyme is highly sensitive to temperature, showing 50% inhibition after incubation for 0.8 min at 45 degrees C or after 23 min at 25 degrees C. It is irreversibly inhibited by disulfiram, and a molar ratio inhibitor: enzyme of 60:1 produced 50% inhibition after incubation for 10 min. A subcellular-distribution study indicates that the enzyme is located in two compartments: the mitochondria, with 60% of the total activity, and the cytosol, with 40% activity. The physiological role of the enzyme in placental amino acid metabolism is discussed.     

Purification of NADH-cytochrome b5 reductase from sheep lung and its electrophoretic, spectral and some other properties

1. NADH-cytochrome b5 reductase was purified from sheep lung microsomes in the presence of non-ionic and ionic detergents, Emulgen 913 and cholate, respectively. 2. The purification procedure involved the ion-exchange chromatography of the detergent solubilized microsomes on DEAE-cellulose. 3. Further purification and concentration of lung reductase was carried out with a second DEAE-cellulose column followed by the affinity column chromatography of partially purified reductase on 5'-ADP-agarose column. 4. The specific activity of sheep lung reductase was 638 mumol ferricyanide reduced/min/mg protein and the yield was 6% of the initial activity in microsomes. 5. The SDS-polyacrylamide gel electrophoresis of the purified lung reductase showed one protein band having the monomer mol. wt of 34,500 +/- 1500. In the presence of 0.4% deoxycholate, it existed as an active dimer having a mol. wt of 68,500. 6. Trypsin treated lung reductase showed two extra protein bands of mol. wts of 28,000 and 25,000 on 10% SDS-polyacrylamide gels. 7. The purified enzyme was found to contain FAD as prosthetic group and the absorption spectrum of lung reductase showed two peaks at 390 and 461 nm which were typical for flavoproteins and a shoulder at 490 nm. 8. The maximal activity of lung reductase was observed between pH 6.5-8.0 and at pH 6.8, when ferricyanide and partially purified sheep lung cytochrome b5 was used as electron acceptors, respectively.     

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase

Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length human mitochondrial 2,4-dienoyl-CoA reductase was previously cloned as pUC18::DECR. PCR methodologies were used to subclone the genes encoding various truncated human mitochondrial 2,4-dienoyl-CoA reductases from pUC18::DECR with primers that were designed to add six continuous histidine codons to the 3' or 5' primer. The PCR products were inserted into pLM1 expression vectors and overexpressed in Escherichia coli. A highly active truncated soluble protein was expressed and purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE. The molecular weight of the protein subunit was 34 kDa. The purified protein is highly stable at room temperature, which makes it potentially valuable for protein crystallization. KM of 26.5 +/- 3.8 microM for 2,4-hexadienoyl-CoA, KM of 6.22 +/- 2.0 microM for 2,4-decadienoyl-CoA, and KM of 60.5 +/- 19.7 microM for NADPH, as well as Vmax of 7.78 +/- 1.08 micromol/min/mg for 2,4-hexadienoyl-CoA and Vmax of 0.74 +/- 0.07 micromol/min/mg for 2,4-decadienoyl-CoA were determined on kinetic study of the purified protein. The one-step purification of the highly active human mitochondrial 2,4-dienoyl-CoA reductase will greatly facilitate further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogs as well as protein crystallization for solving its three-dimensional structure.     

Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.     

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.     

Purification, crystallization, and preliminary x-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.