Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1.

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.

Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1.

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.

Purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase from Streptomyces rochei 303: comparison with an analogous enzyme from Azotobacter sp. strain GP1.

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiol-cleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (kcat/Km = 1.2 and 0.57 s-1.microM-1, respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH2-terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.

Metabolism of 4-chlorophenol by Azotobacter sp. GP1: structure of the meta cleavage product of 4-chlorocatechol.

A mutant strain of Azotobacter sp. GP1 converted 4-chlorophenol to 4-chlorocatechol under cometabolic conditions. Under the same conditions the wild-type strain accumulated a yellow compound, which by chemical and spectroscopic methods was identified as 5-chloro-2-hydroxy-6-oxohexadienoic acid (5-chloro-2-hydroxy-muconic semialdehyde). The structure of this compound indicates a meta-proximal cleavage of 4-chlorocatechol.

Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GP1.

A bacterium which utilizes 2,4,6-trichlorophenol (TCP) as a sole source of carbon and energy was isolated from soil. The bacterium, designated strain GP1, was identified as an Azotobacter sp. TCP was the only chlorinated phenol which supported the growth of the bacterium. Resting cells transformed monochlorophenols, 2,6-dichlorophenol, and 2,3,6-trichlorophenol. Phenol and a number of phenolic compounds, including 4-methylphenol, all of the monohydroxybenzoates, and several dihydroxybenzoates, were very good carbon sources for Azotobacter sp. strain GP1. The organism utilized up to 800 mg of TCP per liter; the lag phase and time for degradation, however, were severely prolonged at TCP concentrations above 500 mg/liter. Repeated additions of 200 mg of TCP per liter led to accelerated degradation, with an optimum value of 100 mg of TCP per liter per h. TCP degradation was significantly faster in shaken than in nonshaken cultures. The optimum temperature for degradation was 25 to 30 degrees C. Induction studies, including treatment of the cells with chloramphenicol prior to TCP or phenol addition, revealed that TCP induced TCP degradation but not phenol degradation and that phenol induced only its own utilization. Per mol of TCP, 3 mol of Cl- was released. 2,6-Dichloro-p-benzoquinone was detected in the resting-cell medium of Azotobacter sp. strain GP1. By chemical mutagenesis, mutants blocked in either TCP degradation or phenol degradation were obtained. No mutant defective in the degradation of both phenols was found, indicating separate pathways for the dissimilation of the compounds. In some of the phenol-deficient mutants, pyrocatechol was found to accumulate, and in some of the TCP-deficient mutants, 2,6-dichlorohydroquinone was found to accumulate.

Serological studies on chloridazon-degrading bacteria.

Agglutination tests and immunofluorescence tests with antisera against four strains of chloridazon-degrading bacteria revealed the serological uniformity of a group of 22 chloridazon-degrading bacterial strains. No serological relationship could be found between chloridazon-degrading bacteria and representatives of other Gram-negative bacteria. This was demonstrated by agglutination tests, including testing of the antiserum against Acinetobacter calcoaceticus, and by immunofluorescence tests, including testing of the sera against Pseudomonas and Acinetobacter strains. The tests were performed with 31 representatives of different Gram-negative bacteria, and with 22 strains of chloridazon-degrading bacteria as antigens. Differences in the extent of agglutination reactions and antibody titres among chloridazon-degrading bacterial strains together with cross-adsorption xperiments, suggest a rough classification of chloridazon-degrading bacteria into two subgroups. On the basis of immunofluorescence data, a linkage-map was worked out to represent serological relationships in the group of chloridazon-degrading strains.

Bacterial conversion of phenylalanine and aromatic carboxylic acids into dihydrodiols.

Strain E of chloridazon-degrading bacteria, when grown on L-phenylalanine accumulates cis-2,3-dihydro-2,3-dihydroxyphenylalanine. In experiments with resting cells and during growth the bacterium converts the aromatic carboxylic acids phenylacetate, phenylpropionate, phenylbutyrate and phenyl-lactate into the corresponding cis-2,3-dihydrodiol compounds. The amino acids L-phenylalanine, N-acetyl-L-phenylalanine and t-butyloxycarbonyl-L-phenylalanine were also transformed into dihydrodiols. All seven dihydrodiols, thus obtained, were characterized both by conventional analytical techniques and by the ability to serve as substrates for a cis-dihydrodiol dehydrogenase.

Acute toxicity testing of some herbicides-, alkaloids-, and antibiotics-metabolizing soil bacteria in the rat.

Seven strains of soil bacteria with the ability to metabolize herbicides, alkaloids or antibiotics were tested in rats for acute toxicity. 1. Upon oral administration of 9.0 x 10(8) to 6.6 x 10(10) cells daily during 7 d no adverse reactions were observed. 2. Exposure by air did not lead to specific pulmonary changes. 3. Intracutaneous injection of 7.5 x 10(6) to 1.4 x 10(8) cells did not lead to adverse skin reactions. 4. Intraperitoneal injections up to 10(8) cells per animal did not kill rats although bacteria entered blood. At higher concentrations some mortality occurred partly due to unspecific stress reactions. 5. Animal data and observations on 20 humans being exposed to these strains for 2 months up to 15 years support the view that the bacteria tested are essentially harmless for health.

[Degradation and biosynthesis of L-phenylalanine by chloridazon-degrading bacteria]. / Abbau und Biosynthese von L-phenylalanin in chloridazon-abbauenden Bakterien.

Incubating chloridazon-degrading bacteria with L-phenylalanine leads to the accumulation of L-2,3-dihydroxyphenylalanine, o-tyrosine and m-tyrosine in the medium. Incubating the bacteria with N-acetyl-L-phenylalanine leads to N-acetyl-(2,3-dihydroxyphenyl)alanine. Using phenylacetic acid as substrate leads to the accumulation of malonic acid. The products are isolated by gel chromatography and high performance liquid chromatography. 2,3-Dihydroxy-L-phenylalanine is attacked by a catechol 2,3-dioxygenase in the presence of Fe2. An unstable yellow compound is formed in this reaction. This meta-cleavage-product is again cleaved by a hydrolase, leading to aspartic acid and 4-hydroxy-2-oxovaleric acid. Both products were isolated fromthe reaction buffer by amino acid analysis and high performance liquid chromatography. The dioxygenase and hydrolase were partially purified and characterized. A new degradation pathway for phenylalanine is discussed and compared with known pathways. The enzymes chorismate mutase, prephenate dehydratase and prephenate dehydrogenase are characterized and inhibition as well as repression are investigated. Only prephenate dehydrogenase is inhibited by phenylalanine, tyrosine and tryptophane. Chorismate mutase is repressed by phenylalanine, prephenate dehydrogenase by phenylalanine and tyrosine. Prephenate dehydratase is not repressed by aromatic amino acids. Regulation of aromatic amino acid biosynthesis in connection with phenylalanine degradation is discussed.

[Purification and properties of two chloridazondihydrodiol dehydrogenases from chloridazon degrading bacteria]. / Reinigung und Eigenschaften von zwei Chloridazondihydrodiol-Dehydrogenasen aus Chloridazon-abbauenden Bakterien.

A cell-free extract of Chloridazon-degrading soil bacteria catalyzes the conversion of the dihydrodiol derivative of chloridazon to the corresponding catechol derivative. NAD is required as hydrogen acceptor. Chromatography of the crude extract on DEAE-cellulose results in the elution of two different enzymes (enzyme A and enzyme B, respectively) with the same catalytic capacity. Both enzymes were purified to homogeneity in disc-gel electrophoresis and their properties were compared. The molecular weight was found to be 220 000 for both enzymes. Dodecyl sulphate polyacrylamide gel electrophoresis indicated subunits of molecular weight 50 000 in both cases. The synthesis of the enzymes does not seem to be under inductive control. The two dehydrogenases differ in heat-stability, pH-optimum, Km-values for the substrate and in their sensitivity to inhibitors. Enzyme A shows relatively high heat lability, a pH-optimum at pH 9.5, and a Km-value of 0.25 mM for the dihydrodiol derivative of chloridazon. The catalytic activity of enzyme A is not influenced by p-chloromercuribenzoate or by N-bromosuccinimide. In contrast enzyme B is relatively stable at high temperatures, showing a pH-optimum of 7.0, and a Km for the dihydrodiol derivative of chloridazon of 1.0 mM. Enzyme B can be completely inhibited by even small amounts of p-chloromercuribenzoate and by N-bromosuccinimide. Striking differences were found in the substrate specificities of the two dehydrogenases. Whereas enzyme A exhibits a high specificity towards dihydrodiols derived from aromates of the chloridazon or phenazon type, enzyme B is much less specific and is also able to convert the dihydrodiols of benzene, toluene or chlorobenzene into the corresponding catechols. Both enzymes are competitively inhibited by the reaction product, the catechol of chloridazon. Other catechols differed in their inhibitory effect on the two dehydrogenases. These differences are correlated with the different substrate specificities.

[Degradation of antipyrin by pyrazon-degrading bacteria (author's transl)]. / Abbau von Antipyrin durch Pyrazon-abbauende Bakterien.

Bacteria with the ability to grow on pyrazon as sole source of carbon were isolated from soil. They also are able to grow on antipyrin. Then three metabolites of antipyrin can be isolated from the culture fluid which were identified as 2,3-dimethyl-1-(cis-2,3-dihydro-2,3-dihydroxy-4,6-cyclohexadiene-1-yl)-pyrazolone (5) (I), as 2,3-dimethyl-1-(2,3-dihydroxyphenyl)-pyrazolone (5) (II) and as 2,3-dimethyl-pyrazolone (5) (III), respectively. Compound I and II were used as substrates for enzyme studies. A dioxygenase catalyzes the enzymatic conversion of antipyrin into compound I. In the presence of NAD as cosubstrate compound I is transformed into compound II by a dehydrogenase. A pure preparation of metapyrocatechase from pyrazon-degrading bacteria converts compound II into the dephenylated heterocyclic moiety of antipyrin (III) and into 2-pyrone-6-carboxylic acid. Based on the results of the enzymatic studies a pathway for the degradation of antipyrin is proposed.

Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria.

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000,red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.

[Enzymatic formation of a cis,cis-muconic acid derivative using pyrazon-degrading bacteria (author's transl)]. / Enzymatische Bildung eines Muconsäurederivates mit Hilfe Pyrazon-abbauender Bakterien

The cis,cis-muconic acid derivative of pyrazon, which was formerly isolated from the medium of pyrazon-degrading bacteria, was formed enzymatically by incubation of the catechol derivative of pyrazon with partially purified ortho pyrocatechase from pyrazon-degrading bacteria.

[Enzymatic formationof 4-hydroxy-2-oxovalerate using pyrazon-degrading bacteria (author's transl)]. / Enzymatische Bildung von 2-Keto-4-hydroxyvaleriansäure mit Hilfe von Pyrazon-abbauenden Bakterien

By treatment of 2-hydroxymuconic acid with a partially purified 4-oxalocrotonate decarboxylase 4-hydroxy-2-oxovalerate could be obtained. Both forms of 4-hydroxy-2-oxovalerate, the keto as well as the enol form could be isolated.

[On the enzymatics of the microbial breakdown of pyrazone (author's transl)]. / Zur Enzymatik des mikrobiellen Pyrazonabbaus

From samples of earth taken in different parts of the world bacteria were isolated which grow on pyrazone as the only source of carbon. When these bacteria are grown in a pyrazone mineral salt medium, four compounds are excreted into the medium. The structures of these compounds furnish information on the catabolic route of pyrazone. Since the suggested scheme of breakdown was incomplete, enzymatic tests were carried out to clarify the matter. It was possible to carry out the first steps of breakdown also in the cell-free extract of the pyrazone-degrading bacteria. For the second step of pyrazone breakdown, 2 different enzymes of the same catalytic activity were identified. For the oxidative cleavage of the pyrocatechole derivative 2 different enzymes were found: an ortho- and a meta-clearing enzyme. The 2-hydroxy muconic acid decarboxylase was identified as a further enzyme. The importance of this enzyme is discussed in connection with the further breakdown.

[Breakdown of the herbicide pyramin by micro-organisms of the soil (author's transl)]. / Der Abbau des Herbizids Pyramin durch Mikroorganismen des Bodens

The herbicide Pyramin, which is employed in the cultivation of beets to combat broad-leaf weeds, contains the herbicidal substance 5-amino-4-chloro-2-phenyl-3 (2H) pyridazinone, abbreviated pyrazone. The breakdown of pyrazone in the soil was investigated and it was found that this substance disappears relatively quickly and that the dephenylated heterocycle of pyrazone 5-amino-4-chloro-3 (2H) pyridazinone is obtained as transformation product. It was possible to isolate bacteria, which grow on pyrazone as the only carbon source, from soil samples originating from different parts of the world. Four compounds are excreted during the cultivation of pyrazone-degrading bacteria in a pyrazone mineral salt medium. With the aid of the structure of these metabolites and enzymatic tests, a scheme for the bacterial breakdown of pyrazone is proposed.