Formaldehyde dehydrogenase from Pseudomonas putida. Purification and some properties

Formaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cell-free extract of Pseudomonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate. The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low. The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria. The enzyme was markedly inhibited by Ni2+, Pd2+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride. The molecular weight of the enzyme was estimated to be 150,000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers. Kinetic analysis gave Km values of 67 microM for formaldehyde and 56 microM for NAD+, and suggested that the reaction proceeds by a "Ping-pong" mechanism. The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.     

Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize gamma-aminobutyraldehyde to gamma-aminobutyric acid.

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5'-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase. It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000. The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed. But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species. The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.     

The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram-positive bacteria Rhodococcus erythropolis. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130 000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross-linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase. A heat-stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofocusing revealed that the cofactor is a weak acid with a pK of about 6.5. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate. It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.     

Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase

Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure.     

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.     

Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase

Formaldehyde dehydrogenase (EC 1.2.1.1) is a widely occurring enzyme which catalyzes the oxidation of S-hydroxymethylglutathione, formed from formaldehyde and glutathione, into S-formyglutathione in the presence of NAD. We determined the amino acid sequences for 5 tryptic peptides (containing altogether 57 amino acids) from electrophoretically homogeneous rat liver formaldehyde dehydrogenase and found that they all were exactly homologous to the sequence of rat liver class III alcohol dehydrogenase (ADH-2). Formaldehyde dehydrogenase was found to be able at high pH values to catalyze the NAD-dependent oxidation of long-chain aliphatic alcohols like n-octanol and 12-hydroxydodecanoate but ethanol was used only at very high substrate concentrations and pyrazole was not inhibitory. The amino acid sequence homology and identical structural and kinetic properties indicate that formaldehyde dehydrogenase and the mammalian class III alcohol dehydrogenases are identical enzymes.     

Purification and properties of the enzyme 6-phosphogluconate dehydrogenase from Dicentrarchus labrax L. liver

The enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ oxidoreductase, decarboxylating EC 1.1.1.44) from bass liver has been purified to over 95% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to about 100,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed to be a dimeric protein. The effect of pH and kinetic properties were studied.     

Substrate specificity of bovine liver formaldehyde dehydrogenase

Formaldehyde dehydrogenases isolated from several different biological sources have been reported to catalyze the NAD+-dependent oxidative acylation of glutathione by methylglyoxal to form S-pyruvylglutathione, suggesting the involvement of this enzyme in the metabolism of methylglyoxal. However, formaldehyde dehydrogenase from bovine liver is found not to use methylglyoxal or related alpha-ketoaldehydes as substrates. Using methylglyoxal with the enzyme under conditions favoring the forward reaction did not result in the formation of S-pyruvylglutathione. Using independently synthesized S-pyruvylglutathione with the enzyme under conditions favoring the reverse reaction did not result in the production of methylglyoxal. In addition, methylglyoxal and several related alpha-ketoaldehydes did not exhibit detectable activity with formaldehyde dehydrogenase partially purified from human liver, contrary to a previous report. Some, if not all, past reports that methylglyoxal serves as a substrate for the dehydrogenase may be due to the demonstrated presence of contaminating formaldehyde in some commercially available preparations of methylglyoxal. In a related study, S-hydroxymethylglutathione, formed by pre-equilibrium addition of formaldehyde to glutathione, is concluded to be direct substrate for the dehydrogenase. This follows from the observation that the catalytic turnover number of the enzyme in the forward direction exceeds by a factor of approximately 20 the first order rate constant for decomposition of S-hydroxymethylglutathione to glutathione and formaldehyde (k = 5.03 +/- 0.30 min-1, pH 8, 25 degrees C).     

Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.     

Purification of a neutral thiol protease from Paragonimus westermani metacercariae by single-step chromatography and preparation of antibodies

A neutral thiol protease from extracts of larvae of the mammalian digenean parasite Paragonimus westermani metacercariae was purified by single-step chromatography on Ultrogel AcA-54, measuring its activity on t-butyloxycarbonyl-valyl-leucyl-lysyl-4-methylcoumaryl-7-amide as a substrate. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and size exclusion-high-performance liquid chromatography analysis of the enzyme indicated that the fraction obtained by gel filtration was homogeneous. Antibodies against the purified protease were raised in rabbits by immunizing with micro quantities of the enzyme protein. The antibodies revealed a single precipitin line against the enzyme on double immunodiffusion analysis.     

Purification and Characterization of Formaldehyde Dehydrogenase in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

An NAD-linked formaldehyde dehydrogenating enzyme was found in the cell-extract of Kloeckera sp. No. 2201, which utilized methanol as a sole source of carbon. The enzyme was inducibly formed in methanol-grown cells. This fact suggests that the enzyme may play a significant role in the methanol metabolism of this yeast. The enzyme was purified from a cell-extract by ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and on hydroxylapatite, and Sephadex G-200 gel filtration. From an experiment with the purified enzyme, it was found that the enzyme specifically required reduced glutathione for activity, and was reactive toward methylglyoxal as well as formaldehyde. The enzyme catalyzed the following reaction:

the enzyme was concluded to be a kind of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1). Other properties of the enzyme were also investigated.     

Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms

1. Adult Clonorchis sinensis, the Chinese liver fluke, is known to migrate to the bile ducts of its mammalian host and cause significant pathology. 2. An acidic, thiol-dependent proteinase with a native mol. wt of approximately 18,500 was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. By SDS-polyacrylamide gel electrophoresis, the mol. wt of the enzyme was estimated to be 15,000. 3. The enzyme was similar to cathepsin B-like cysteine proteinases based on pH optimum, substrate specificity, and inhibitor sensitivity. 4. Antisera from human clonorchiasis and C. sinensis-infected rabbits reacted in immunoblots with the partially purified proteinase. The C. sinensis proteinase may be useful for serodiagnosis of clonorchiasis.     

Purification and properties of aldehyde dehydrogenase from Saccharomyces cerevisiae.

A procedure for the purification of aldehyde dehydrogenase from bakers' yeast (Saccharomyces cerevisiae) is reported. Treatment with acid, heat and organic solvents was avoided and chromatographic and filtration techniques in the presence of phenylmethylsulfonylfluoride were mainly used. An affinity chromatography step using the reactive dye Cibacron blue F3G-A, which was covalently bound to Sepharose 4B, was found to be essential. The enzyme was bound to and then released from the dye. The purified enzyme was shown to be homogeneous by gel filtration, disc electrophoresis and SDS electrophoresis. The molecular weight of the purified enzyme determined by gel filtration was 170,000, which agreed with that of the enzyme in the crude extract. The enzyme was composed of subunits of a molecular weight of 57,000. The specific activity of the enzyme was 20 units per mg of protein under the standard assay conditions. The substrate specificity, the relative maximal velocity, the michaelis constants, the pH optimum, the stability and the activation energy of the enzyme are reported.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Purification and properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium

An intracellular aryl-alcohol dehydrogenase (previously referred to as aryl-aldehyde reductase) was purified from the white-rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy-substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4-benzyloxy-3-methoxybenzaldehyde). The highest reduction rate was measured when 3,5-dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47-kDa protein were able to immunoprecipitate the aryl-alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl-alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.     

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.     

NADP-dependent isocitrate dehydrogenase from bass (Dicentrarchus labrax L.) liver

The isocitrate dehydrogenase from bass liver was purified to homogeneity by gel filtration, affinity and ion exchange chromatographies. The molecular weight was estimated by gel filtration chromatography to about 120,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed it to be a dimeric protein. The enzyme showed maximum activity in the pH range between 7.0 and 8.0 while its maximum activity was at pH 7.5. DL-Isocitrate and Mn2+ stabilized the enzyme, while NADP had the opposite effect. The Km for isocitrate was 0.31 mM and the Km for NADP was 36 microM.     

Purification and characterization of a processing protease from rat liver mitochondria.

A processing protease has been purified from the matrix fraction of rat liver mitochondria. The purified protease contained two protein subunits of 55 kd (P-55) and 52 kd (P-52) as determined by SDS-PAGE. The processing protease was estimated to be 105 kd in gel filtration, indicating that the two protein subunits form a heterodimeric complex. At high ionic conditions, the two subunits dissociated. The purified processing protease cleaved several mitochondrial protein precursors destined to different mitochondrial compartments, including adrenodoxin, malate dehydrogenase, P-450(SCC) and P-450(11 beta), but the processing efficiencies were different each other. The endoprotease nature of the processing protease was confirmed with the purified enzyme using adrenodoxin precursor as the substrate; both the mature form and the extension peptide were detected after the processing. The processing activity of the protease was inhibited by metal chelators, and reactivated by Mn2+, indicating that the protease is a metalloprotease.     

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.