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 properties of carnitine dehydrogenase from Pseudomonas putida

Carnitine dehydrogenase (carnitine:NAD+ oxidoreductase, EC 1.1.1.108) from Pseudomonas putida IFP 206 catalyzes the oxidation of L-carnitine to 3-dehydrocarnitine. The enzyme was purified 72-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The molecular mass of this enzyme is 62 kDa and consists of two identical subunits. The isoelectric point was found to be 4.7. the carnitine dehydrogenase is specific for L-carnitine and NAD+. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0 and 7.0 in the reduction reaction. The optimal temperature is 30 degrees C. The Km values for substrates were determined.     

Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida.

The purification of (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase from cells of Pseudomonas putida grown with toluene as the sole source of carbon and energy is reported. The molecular weight of the enzyme is 104,000 at pH 9.7. The enzyme is composed of four apparently identical subunits with molecular weights of 27,000. The enzyme is specific for nicotinamide adenine dinucleotide and oxidizes a number of cis-dihydrodiols. Both enantiomers of a racemic mixture of cis-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol are oxidized by the enzyme. No enzymatic activity is observed with trans-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol.     

Purification and characterization of a novel enzyme, N-carbamoylsarcosine amidohydrolase, from Pseudomonas putida 77

N-Carbamoylsarcosine amidohydrolase, a novel enzyme involved in the microbial degradation of creatinine in Pseudomonas putida 77, was purified 27-fold to homogeneity with a 63% overall recovery through simple purification procedures including successive ammonium sulfate fractionation, DEAE-cellulose chromatography, and crystallization. The relative molecular mass of the native enzyme estimated by the ultracentrifugal equilibrium method is 102,000 +/- 5000, and the subunit Mr is 27,000. The Km and Vm values for N-carbamoylsarcosine are 3.2 mM and 1.75 units/mg protein, respectively. Ammonia, carbon dioxide, and sarcosine were formed stoichiometrically from N-carbamoylsarcosine through the action of the purified enzyme preparation. N-Carbamoyl amino acids with a methyl group or hydrogen atom on the amino-N atom and possessing glycine, D-alanine, or one of their derivatives as an amino acid moiety served well as substrates for N-carbamoylsarcosine amidohydrolase. N-Carbamoylsarcosine, N-methyl-N-carbamoyl-D-alanine, N-carbamoylglycine, and N-carbamoyl-D-alanine were hydrolyzed at relative rates of 100, 12.8, 9.8, and 7.3, respectively, by the enzyme. N-Carbamoyl derivatives of D-tryptophan, D-phenylalanine, and those of some other amino acids including D-phenylglycine and p-hydroxy-D-phenylglycine were also hydrolyzed by the enzyme. For the L-isomers of all N-carbamoyl amino acids tested there was no production of ammonia, carbon dioxide, or the corresponding amino acids due to the action of the enzyme. Cupric, mercuric, and silver ions inhibited the enzyme strongly, and some thiol reagents were also found to be inhibitory.     

Purification and characterization of methioninase from Pseudomonas putida.

Methioninase of Pseudomonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract. 1. The purified enzyme had an S20,w of 8.37, a molecular weight of 160,000, and an isoelectric point of 5.6. 2. A break in the Arrhenius plot was observed at 40 degrees and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively. 3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of alpha-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 muM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).     

Purification, crystallization, and some properties of creatine amidinohydrolase from Pseudomonas putida.

A method was developed for purification and crystallization of creatinase [creatine amidinohydrolase, EC 3.5.3.3] from Pseudomonas putida var. naraensis C-83. The purified preparation appeared homogeneous on disc electrophoresis and ultracentrifugation and had a molecular weight of 94,000. It was most active at pH 8 and stable between pH 6 and 8 for 24 hr at 37 degrees. SDS-polyacrylamide gel electrophoresis indicated that the native enzyme was made up of two subunit monomers, the molecular weights of which were estimated to be 47,000. Inhibition experiments suggested that a sulfhydryl group is located in or near the active site of the enzyme.     

Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40.

The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented. The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes. Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment. However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum. The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride. Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000. Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors. An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating. Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme.     

Purification and properties of pyrocatechase II from Pseudomonas putida strain 87]

Induction of modified ortho-pathway enzymes (catechol 1.2-dioxygenase II, muconate cycloisomerase II, dienelactone hydrolase, and maleylacetate reductase) was found in Pseudomonas putida 87, when 3-chlorobenzoic acid was used as a sole carbon and energy source. Catechol 1.2-dioxygenase II, the key chlorocatechol cleaving enzyme, was purified and characterized. The enzyme molecular mass as determined by gel filtration was 65,000 Da; the minimum molecular mass upon SDS electrophoresis was 33,000 Da. The pH and temperature optima for the enzyme were 7.2-7.8 and 35 degrees C, respectively. The highest stability of catechol 1.2-dioxygenase II upon storage was observed in 50 mM Tris-HCl buffer pH 7.8 at 4 degrees C. The relative values of Vmax for catechol 1.2-dioxygenase II with 3-chloro-, 4-chloro-, and 3.5-dichlorocatechols were 28%, 50%, and 41% of those for catechol. The enzyme affinity for chlorocatechols was 3-9 times higher than for methylcatechols and 10-20 times higher than for unsubstituted catechol.     

Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Pseudomonas putida by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2 x 10(-4) M and 5.9 x 10(-5) M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.     

Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2

1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH(4))(2)SO(4) fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Delta(2)-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27x10(6) and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5-9.5, a K(m) for 2-furoyl-CoA of 20.2mum and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.     

Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1

Toluene dioxygenase oxidizes toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. This reaction is catalyzed by a multienzyme system that is induced in cells of Pseudomonas putida F1 during growth on toluene. One of the components of toluene dioxygenase has been purified to homogeneity and shown to be an iron-sulfur protein that has been designated ferredoxinTOL. The molecular weight of ferredoxinTOL was calculated to be 15,300, and the purified protein was shown to contain 2 g of atoms each of iron- and acid-labile sulfur which appear to be organized as a single [2Fe-2S]cluster. Solutions of ferredoxinTOL were brown in color and showed absorption maxima at 277, 327, and 460 nm. A shoulder in the spectrum of the oxidized protein was discernible at 575 nm. Reduction with sodium dithionite or NADH and ferredoxinTOL reductase resulted in a decrease in visible absorbance at 460 and 575 nm, with a concomitant shift in absorption maxima to 382 and 438 nm. The redox potential of ferredoxinTOL was estimated to be -109 mV. In the oxidized state, the protein is diamagnetic. However, upon reduction it exhibited prominent electron paramagnetic resonance signals with anisotropy in g values (gx = 1.81, gy = 1.86, and gz = 2.01). Anaerobic reductive titrations revealed that ferredoxinTOL is a one-electron carrier that accepts electrons from NADH in a reaction that is mediated by a flavoprotein (ferredoxinTOL reductase). The latter is the first component in the toluene dioxygenase system. Reduced ferredoxinTOL can transfer electrons to cytochrome c or to a terminal iron-sulfur dioxygenase (ISP-TOL) which catalyzes the incorporation of molecular oxygen into toluene and related aromatic substrates.     

Purification and some properties of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

Human liver 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was purified 17 500-fold to apparent homogeneity as judged from polyacrylamide-gel disc electrophoresis. A pH optimum of 7.7-9.0 was found. The Km value was pH- and temperature-dependent. At 37 degrees C and pH 7.7, Km was 0.16 mM and it increased to 0.29 at pH 6.0 and 0.23 at pH 9.0. At 25 degrees C and pH 7.7, a Km value of 0.99 mM was obtained. When the substrate concentration was varied, apparent Michaelis-Menten kinetics were obtained. p-Hydroxymercuribenzoate, glutathione or cysteine had no effect on the enzyme activity; 5 mM-N-acetylcysteine inhibited about 47% of the total enzyme activity. Apart from Cu2+, other bivalent ions were virtually ineffective at 1 mM. The kinetic study differentiates this enzyme from aspartylglucosylaminase from other sources.     

Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens

In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion. Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth. In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells. Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures. Exopolysaccharide from both strains was isolated and purified. Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P. fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively. Polymers from both strains were acetylated to a variable degree.     

Purification and characterization of glyoxalase I from Pseudomonas putida

Glyoxalase I was purified to apparent homogeneity from Pseudomonas putida. The enzyme was a monomer with a molecular weight of 20,000. The enzyme was most active at pH 8.0. The Km values for methylglyoxal and 4,5-dioxovale-rate are 3.5 mM and 1.2 mM, respectively. Contrary to the case of eukaryotic enzymes, chelating agents showed little inhibitory effects on the enzyme activity. Among the metal ions tested, Zn++ specifically and completely inhibited the activity of the enzyme at a millimolar level. The properties of bacterial glyoxalase I were quite different from mammalian and yeast enzymes.