Hydroxylation of quinaldic acid: quinaldic acid 4-monooxygenase from Alcaligenes sp. F-2 versus quinaldic acid 4-oxidoreductases.

The N-heterocycles quinaldic acid (quinoline 2-carboxylic acid), kynurenic acid (4-hydroxyquinoline 2-carboxylic acid), 2-oxo-1,2-dihydroquinoline, and xanthine are utilized by Alcaligenes sp. F-2 as sole source of carbon and energy. Although quinoline did not serve as growth substrate, 8-hydroxy-2-oxo-1,2-dihydroquinoline and 8-hydroxycoumarin, metabolites of the 'coumarin pathway' of quinoline catabolism, were isolated from the culture fluid during growth on 2-oxo-1,2-dihydroquinoline. Contrary to Serratia marcescens 2CC-1 and Pseudomonas sp. AK-2 (Sauter et al. (1993) Biol. Chem. Hoppe-Seyler 374, 1037-1046), which possess different molybdenum-containing hydroxylases catalysing the 4-hydroxylation of quinaldic acid to kynurenic acid with incorporation of oxygen derived from water and concomitant reduction of an electron acceptor, Alcaligenes sp. F-2 contains an inducible quinaldic acid 4-monooxygenase that catalyses the very same conversion in the presence of O2 and NADH. The activity of the monooxygenase was enhanced 1.5-fold by Fe2+ ions. The extremely thermolabile enzyme (apparent molecular mass: 155 kDa) exclusively accepted quinaldic acid as substrate. The 'pseudosubstrates' menadione, 8-hydroxyquinoline, and 8-hydroxy-2-oxo-1,2-dihydroquinoline effected consumption of NADH and oxygen without being hydroxylated. Quinaldic acid 4-monooxygenase was inhibited by sulfhydryl modifying and chelating agents, and by various divalent metal ions, whereas reducing agents did not affect enzymatic activity.

The reaction of the mutagen 1,1'-hexamethylene-bis-[(5-p-chlorophenyl)-biguanide] with guanosine and cysteine.

The mutagen 1,1'-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] reacts at 37 degrees C with guanosine and guanine to yield xanthosine or xanthine and oxidizes cysteine to cystine. After treatment of a guanosine-labelled DNA sample from Escherichia coli with the mutagen xanthine could be detected as a reaction product. At a slow rate the mutagen is hydrolysed spontaneously yielding urea, 1.6-hexanediol and 4-chloroaniline. The reaction mechanisms both of the hydrolysis and of the reaction with cysteine and guanosine are discussed.

The 2Fe2S centres of the 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy.

The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity. We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system. The oxygenase component was identified as a Rieske [2Fe2S] protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gav = 1.893. The reductase component, an iron-sulfur flavoprotein, contained a [2Fe2S] cluster with gz,y,x = 2.03, 1.94, 1.89 and the average g-value (gav) of 1.953, typical of a ferredoxin-type centre. In redox titrations at pH 7, the midpoint potentials were determined to be -180 mV +/- 30 mV and -100 mV +/- 10 mV for the reductase and oxygenase component, respectively. A detailed comparison to other multicomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved.

Cloning, sequence analysis, and expression of the Pseudomonas putida 33/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase gene, encoding a carbon monoxide forming dioxygenase.

1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from the 1H-4-oxoquinoline utilizing Pseudomonas putida strain 33/1, which catalyzes the cleavage of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilate, is devoid of any transition metal ion or other cofactor and thus represents a novel type of ring-cleavage dioxygenase. Gene qdo was cloned and sequenced. Its overexpression in Escherichia coli yielded recombinant His-tagged Qdo which was catalytically active. Qdo exhibited 36% and 16% amino acid identity to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) and atropinesterase (a serine hydrolase), respectively. Qdo as well as Hod possesses a SXSHG motif, resembling the motif GXSXG of the serine hydrolases which comprises the active-site nucleophile (X=arbitrary residue).

Crystallization and preliminary X-ray data of bromoperoxidase from Streptomyces aureofaciens ATCC 10762.

Bromoperoxidase from Streptomyces aureofaciens ATCC 10762, a non-haem haloperoxidase, has been crystallized using the hanging drop method. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group P2(1)3 with a = 123.4 A. The asymmetric unit contains a dimer of Mr = 60,200. The crystals diffract to at least 2.3 A resolution and are suitable for crystallographic structure analysis.

Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia: sequence, expression in heterologous hosts, and purification of the enzyme.

The nucleotide sequence of a 1.5-kb fragment of Pseudomonas pyrrocinia DNA containing the chloroperoxidase(CPO)-encoding gene (cpo) and its flanking regions was determined. The cpo codes for a protein of 278 amino acids (aa). The mature enzyme contains no N-terminal methionine, so that the CPO monomer consists of 277 aa with a calculated M(r) of 30,304. Expression studies showed that the cpo from P. pyrrocinia is functionally expressed in Escherichia coli and Streptomyces lividans. Based on the overproduction of the CPO in E. coli, a novel and simple purification procedure was developed allowing the isolation of about 800-fold more CPO per gram of cells than was originally isolated from P. pyrrocinia. Comparison with the aa sequence of the bromoperoxidase BPO-A2 from S. aureofaciens ATCC10762 revealed an identity of 38%.

Detection of a bromoperoxidase in Streptomyces phaeochromogenes.

A bromoperoxidase could be detected after fractionation in the chloramphenicol producing actinomycete, Streptomyces phaeochromogenes. This enzyme is capable of catalyzing the bromination of the antifungal antibiotic pyrrolnitrin [3-chloro-4-(2-nitro-3-chlorophenyl)pyrrole] in the 2-position of the pyrrole ring. The enzyme had a pH optimum of 5.0. This procaryotic bromoperoxidase requires the presence of H2O2 and can also brominate monochlorodimedone, but cannot catalyze chlorination. This enzyme is the first haloperoxidase described from procaryotic sources.

Cloning and high-level expression of a chloroperoxidase gene from Pseudomonas pyrrocinia in Escherichia coli.

A chloroperoxidase gene from Pseudomonas pyrrocinia was cloned into Escherichia coli using the cosmid vector pJB8. The gene coding for the chloroperoxidase could be localized to a 1.5 kb fragment of DNA which was subcloned into the high-copy-number plasmid pUC18. In one subclone increased halogenating activity could be found which was 570-fold greater than in P. pyrrocinia. The halogenating enzyme was identified as the chloroperoxidase by SDS-polyacrylamide gel electrophoresis.

Detection of a new chloroperoxidase in Pseudomonas pyrrocinia.

A new chloroperoxidase could be detected in Pseudomonas pyrrocinia ATCC 15,958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. This enzyme was separated from a ferriprotoporphyrin IX containing bromoperoxidase which was also produced by this bacterium. The enzyme is capable of catalyzing the chorination of indole to 7-chloroindole. This procaryotic chloroperoxidase requires the presence of H2O2 and can also brominate monochlorodimedone, but cannot catalyze its chlorination. This enzyme is the first chloroperoxidase described from procaryotic sources.

Purification of arogenate dehydrogenase from Phenylobacterium immobile.

Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.

Degradation of quinoline-4-carboxylic acid by Microbacterium sp.

From soil enrichment culture of quinoline-4-carboxylic acid-degrading bacterium was isolated. The organism was identified as Microbacterium sp. Mutants were induced with N-methyl-N'-nitro-N-nitrosoguanidine. One mutant accumulated successively two metabolites which were identified as 2-oxo-1,2-dihydro-quinoline-4-carboxylic acid and 8-hydroxy-2-oxo-2H-1-benzopyran-4-carboxylic acid.

Transformation of 2-chloroquinoline to 2-chloro-cis-7,8-dihydro-7,8- dihydroxyquinoline by quinoline-grown resting cells of Pseudomonas putida 86.

Resting cells of Pseudomonas putida strain 86 were grown on quinoline transformed 2-chloroquinoline to 2-chloro-cis-7,8-dihydro-7,8-dihydroxyquinoline which was not converted further. 7,8-Dioxygenating activity was present when the enzymes of quinoline catabolism were induced. Quinoline-grown cells of strain 86 treated simultaneously with 2-chloroquinoline and D-(-)-threo-chloramphenicol to prevent protein biosynthesis also formed the cis-7,8-dihydrodiol of 2-chloroquinoline. Succinate-grown resting cells did not oxidize 2-chloroquinoline. Acid-catalyzed decomposition of 2-chloro-cis-7,8-dihydro-7,8-dihydroxyquinoline predominantly yielded 2-chloro-8-hydroxyquinoline. By analogy, accumulation of the putative dead-end metabolite 1H-8-hydroxy-2-oxoquinoline during growth of P. putida 86 on quinoline is suggested to likewise result from dehydration of the 7,8-dihydrodiol of 1H-2-oxoquinoline.

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 aromatic carboxylic acids by acinetobacter.

The degradation of the aromatic carboxylic acids 3-phenylpropionic acid, cinnamic acid and L-phenylalanine was investigated in several strains of Acinetobacter calcoaceticus var. lwoffii. Evidence is presented for the conversion of 3-phenylpropionic acid into the cis-2,3-dihydrodihydroxy-derivative, which is further metabolized to 3-(2,3-dihydroxyphenyl-)propionic acid, followed by cleavage of the aromatic ring in meta-proximal position. When pre-grown on 3-phenylpropionic acid, cinnamic acid as well as L-phenylalanine were metabolized under resting cell conditions via the same degradation pathway. When cultivated with cinnamic acid as sole carbon source 4-hydroxycinnamate, together with 4-hydroxybenzoate and protocatechuate were found as metabolites. L-phenylalanine, when provided as the only carbon source, is converted via phenylacetic acid to 2-hydroxyphenyl-acetic acid. All metabolites were identified by conventional chemical techniques. Enzymatic studies yielded further support for the proposed pathways. In 14 of 15 Acinetobacter strains the presence of plasmid DNA could be detected. The number of plasmids varied between 1 and 7. Regarding the number and size of the plasmids considerable variations within the different bacterial strains were observed. No extended homology among the plasmids could be shown by restriction endonuclease digestion. One strain exhibiting a high spontaneous mutation rate to the 3-phenylpropionic acid-negative phenotype did not show any change in its plasmid pattern. The results lead us to conclude that a correlation between the degradative properties of these strains and the presence of plasmid DNA is unlikely.

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.

Aromatic Amino Acid Biosynthesis in a 4-Chlorobenzoic Acid Degrading Pseudomonas Species; Phenylalanine and Tyrosine Synthesis via Arogenate.

The aromatic amino acid biosynthesis was studied in Pseudomonas sp. strain CBS 3 which is able to grow on 4-chlorobenzoic acid. The regulation patterns of anthranilate synthase, chorismate mutase, prephenate dehydratase, prephenate dehydrogenase, arogenate dehydratase and arogenate dehydrogenase were established. The synthesis of anthranilate synthase was induced by L-phenylalanine and L-tyrosine. The enzyme activity was inhibited by L-tryptophan. Chorismate mutase was neither repressed nor inhibited by phenylalanine and tyrosine, but induced by tryptophan. Prephenate dehydratase was both induced and activated by L-tyrosine. The prephenate dehydrogenase activity was inhibited by tyrosine. Arogenate, the transamination product of prephenate, was assayed as a substrate in a crude enzyme preparation. Both enzymes, arogenate dehydratase and arogenate dehydrogenase, could be identified in a crude enzyme preparation. Arogenate dehydrogenase showed the same regulatory pattern as prephenate dehydrogenase.

Mutagenic effect of 1.1'-hexamethylene-bis-[(5-p-chlorophenyl)-biguanide].

The strongly effective bactericidal compound 1.1'-hexamethylene-bis-[(5-p-chlorophenyl)-biguanide] (HCG) induces mutations with a slight inactivation rate in the auxotrophic strains Salmonella typhimurium TA 1535 and TA 1538 in 0.4 microM solution. The mutagenic effect could be confirmed by using the plate incorporation test and the repair test. As phenylethylbiguanide at different inactivation rates does not show any mutagenic effect, the biguanide structure does not seem to be responsible for chemical mutagenesis. A hypothetic mutation mechanism is proposed and compared with the corresponding reaction mechanism of N-methyl-N'-nitro-N-nitro-soguanidine (MNNG).

Effects of 1,1'-hexamethylene-bis-[(5-p-chlorophenyl)-biguanide] on the genome and on the synthesis of nucleic acids and proteins in the bacterial cells.

The strongly effective bactericidal compound 1,1'-hexamethylene-bis-[(5-p-chlorophenyl)-biguanide] (HCG), which is used as a disinfectant alterates the DNA of B. subtilis as shown in the rec assay, induced auxotrophic mutants in E. coli B and causes prophage induction in Micrococcus lysodeikticus 53-40 (N5). In vivo experiments with E. coli B have demonstrated that HCG extensively breaks down bacterial DNA and interacts with the synthesis of cellular DNA to the similar extent as found for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The structural integrity of ribosomes and of ribosomal subunits remains intact in the presence of HCG.

Mutagenic actions of chlorocholine chloride.

Chlorocholine chloride, at concentrations of 0.2 to 0.5 M (3.2 to 7.9%) between pH 5 and 8, showed no significant mutagenic effect whereas at the same concentrations at pH 9 it caused mutations, in a valine-sensitive strain of E. coli K12. Treatment of E. coli B with chlorocholine chloride at 0.5 M and pH 9 resulted in auxotrophic and regulatory deficient (valine-sensitive) mutants. The mutagenic effects of chlorocholine chloride were compared with the effects caused by the food additive NaHSO3.

Mutagenic and inactivating effects of methyl alkylaminosulfonates on Escherichia coli.

Methyl alkylamino methanesulfonates are mutagenic agents as shown by treating several strains of E. coli at pH 7. Methyl methylaminosulfonate (CH3-NH-SO3-CH3) was more efficient than methyl ethylaminosulfonate (C2H5-NH-SO3-CH3) which itself was more efficient than methyl isopropylaminosulfonate (C3H7-NH-SO3-CH3). Methyl methylaminosulfonate seemed to be at least as effective as methyl methanesulfonate (CH3-SO3-CH3). Methyl methylaminosulfonate produced a yield of up to 1% of auxotrophic mutants. All three new mutagens appeared to react according to the same mechanism by ester fission and methylation of nucleophilic groups as is known for methyl methanesulfonate. The reaction mechanism seems to be of the SN2 type.