Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both M?ssbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.     

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 microM tungstate contained a benzylviologen-linked aldehyde oxidoreductase. The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000. It contained 0.68 +/- 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit. After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained. Acetaldehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent Km values of 12.5, 10.8, and 20 microM, respectively. The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent Km, 0.55 mM). The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate. In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97. The dithionite-reduced enzyme exhibited a broad signal at low temperature with g = 2.04 and 1.92; this is indicative of a [4Fe-4S]1+ cluster interacting with a second paramagnet, possibly the S = 1 system of W(IV). Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea. The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.     

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.     

Purification and characterization of NAD-dependent isocitrate dehydrogenase from Dictyostelium discoideum

Isocitrate dehydrogenase (threo-d_s-isocitrate: NAD oxidoreductase (decar☐ylating) EC 1.1.1.41) from Dictyostelium dicoideum was purified 161-fold. The purified enzyme was NAD specific and required Mn²⁺ for activity. Isocitrate consumption and 2-oxoglutarate and NADH production were stoichiometric; no NADH oxidase or glutamate dehydrogenase activities were detected. The pH optimum range for activity was pH 7.5–8.5. Reductive car☐ylation of 2-oxoglutarate with NADH could not be demonstrated. Lineweaver - Burk plots of data from initial velocity studies were linear. There was no evidence of allosteric control by reported effectors (AMP, ADP, citrate) of isocitrate dehydrogenase activity. The reaction was inhibited by NADH. The inhibition by NADH was competitive when either isocitrate or NAD was the variable substrate. 2-Oxoglutarate was not inhibitory at concentrations below 4 mm. The Michaelis constant (K_m) and dissociation constant (K_ib) for isocitrate were 0.16 mm; and K_m and dissociation constant (K_ia) for NAD were 0.34 mm. The inhibition constant for NADH was 0.02 mm. The data are consistent with a rapid equilibrium random bi-bi reaction mechanism (Cleland nomenclature). The NAD-linked isocitrate dehydrogenase activity was also demonstrated in crude extracts of isolated mitochondria.     

Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas.

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.     

Isolation and characterization of an ornithine-containing lipid from Desulfovibrio gigas.

The isolation and characterization of an ornithine-containing lipid obtained from Desulfovibrio gigas are reported. The general structure for this aminolipid is represented by NH2-CH2-(CH)2-CHNH(CO-CH2CH(O-COR2)-R1)-COOH, where R1 represents 3-hydroxy palmitate linked through an amide bond to the alpha-amino group of ornithine, and R2 represents a complex variety of fatty acids esterified to the hydroxyl group of 3-hydroxy palmitate. Fatty acids characterized were n-C14:0 (21%), iso-C14:0 (14%) anteiso-C15:0 (43%), n-C16:0 (2%), n-C18:0 (8%), and n-C 18:1 (11%). The quantitative relationships between aminolipid and phospholipids showed the aminolipid to represent the major polar lipid. Isolation of the cytoplasmic and outer membranes of D. gigas showed the aminolipid to be evenly distributed between both membrane fractions, suggesting a compensatory role in phospholipid-deficient membranes.     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas.

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at - 15 degrees C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its Km value for thiosulfate was calculated to be 5 - 10(-4)M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c3 (Mr = 13000) and c3 (Mr = 26000).     

Purification and partial characterization of alcohol dehydrogenase from wheat.

Further support for hypotheses proposed earlier for the genetic control and subunit composition of the alcohol dehydrogenase of Triticum has been obtained through the purification and partial characterization of the enzyme. The alcohol dehydrogenase of the wheat T. monococcum was purified 103-fold to a specific activity of 55,900 units/mg. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion-exchange chromatography, and preparative isoelectric focusing. The native enzyme has a molecular weight of 116,000 and a dimeric subunit structure. The apparent Michaelis constants are 1.2 × 10^?2m for ethanol and 1 × 10^?4m for NAD. The substrate specificity of wheat alcohol dehydrogenase differs significantly from the substrate specificities of the enzymes of horse and yeast.     

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough

Abstract Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83 500 Da) is proposed to contain a molybdenum cofactor, a 27 000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14000 Da) holds a c -type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.     

Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas

The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at-15°C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220 000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its K _m value for thiosulfate was calculated to be 5·10^-4 M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c ₃ (M _r =13000) and c ₃ (M _r =26000).     

Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.     

Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures

An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a M_r of ca. 77,000. Each subunit with a M_r of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.Abbreviations ADH alcohol dehydrogenase - DTT dithiothreitol - PMSF dephenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - IAA indole-3-acetic acid - TFA trifluoroacetic acid     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Purification and preliminary characterization of alcohol dehydrogenase from Aspergillus nidulans.

Aspergillus alcohol dehydrogenase is produced in response to growth in the presence of a wide variety of inducers, of which the most effective are short-chain alcohols and ketones, e.g. butan-2-one and propan-2-ol. The enzyme can be readily extracted from fresh or freeze-dried cells and purified to homogeneity on Blue Sepharose in a single step by using specific elution with NAD+ and pyrazole. The pure enzyme has Mr 290 000 by electrophoresis or gel filtration; it is a homopolymer with subunit Mr 37 500 by electrophoresis in sodium dodecyl sulphate; its amino acid composition corresponds to Mr 37 900, and the native enzyme contains one zinc atom per subunit. The enzyme is NAD-specific and has a wide substrate activity in the forward and reverse reactions; its activity profile is not identical with those of other alcohol dehydrogenases.     

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures.

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 mumol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecylsulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immuno-reactive 40-kD protein.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum.

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).     

Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad.

Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol.     

Purification and characterization of an NAD-dependent malate dehydrogenase from leaves of the crassulacean acid metabolism plant Aptenia cordifolia

An NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) was purified and characterized from leaves of Aptenia cordifolia L. f. (Schwant). This plant performs crassulacean acid metabolism (CAM), as indicated by: (a) elevated levels of phosphoenolpyruvate carboxylase (PEPC) and NAD(P) malic enzyme; (b) regulation of PEPC compatible with its function during the night; (c) characteristic day/night changes in titratable acidity; and (d) gas exchange profile consistent with that shown by CAM plants. These features remained unchanged by water availability or salt stress, suggesting constitutive CAM. The purified MDH showed a subunit molecular mass of 39.4 kDa, a native mass of 83 kDa (dimer) and a pI of 5.8. It cross-reacted with antibodies against cytosolic malate dehydrogenase (cMDH) from pineapple. Maximum activities for oxaloacetate (OAA) reduction or malate oxidation were observed at pH 7.0 and between pH 7.2 and 8.4, respectively. The enzyme was inhibited by excess OAA, in a pH-dependent manner. A discontinuity was observed in Arrhenius plots at 33 °C, with an activation energy twice as high below this temperature. Although immunologically related, some physical and kinetic dissimilarities between the A. cordifolia and pineapple enzymes suggest that diverse CAM metabolic subtypes may require different MDH isozymes to carry out OAA reduction.