hydroxymuconic-semialdehyde and catechol

The ability of Pseudomonas fluorescens 26K strain to utilize naphthalene at concentrations up to 600 mg/liter as the sole source of carbon and energy in mineral liquid media was shown. Using HPLC, TLC, and mass-spectrometry, the intermediates of naphthalene transformation by this strain were identified as naphthalene cis-1,2-dihydrodiol, salicylaldehyde, salicylate, catechol, 2-hydroxymuconic semialdehyde, and 1-naphthol. Catechol 2,3-dioxygenase (a homotetramer with native molecular mass 125 kDa) and NAD+-dependent homohexameric naphthalene cis-1,2-dihydrodiol dehydrogenase with native molecular mass 160 kDa were purified from crude extract of the strain and characterized. NAD+-dependent homodimeric salicylaldehyde dehydrogenase with molecular mass 110 kDa was purified and characterized for the first time. Based on the data, a pathway of naphthalene degradation by P. fluorescens 26K is suggested.

Topics: Aldehyde Oxidoreductases; Aldehydes; Catechol 2,3-Dioxygenase; Catechols; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Unsaturated; Mass Spectrometry; Naphthalenes; Naphthols; Oxidoreductases Acting on CH-CH Group Donors; Pseudomonas fluorescens; Salicylates

The catechol meta cleavage pathway is one of the central metabolic pathways for the degradation of aromatic compounds. A novel organization of the pathway genes, different from that of classical soil microorganisms, has been observed in Sphingomonas sp HV3 and Pseudomonas sp. DJ77. In a Comamonas sp. JS765, cdoE encoding catechol 2,3-dioxygenase shares a common ancestry only with tdnC of a Pseudomonas putida strain, while codG encoding 2-hydroxymuconic semialdehyde dehydrogenase shows a higher degree of similarity to those genes in classical bacteria. Located between cdoE and cdoG are several putative genes, whose functions are unknown. These genes are not found in meta pathway operons of other microorganisms with the exception of cdoX2, which is similar to cmpX in strain HV3. Therefore, the gene cluster in JS765 reveals a third type of gene organization of the meta pathway.

Topics: Amino Acid Sequence; Bacterial Proteins; Biodegradation, Environmental; Catechol 2,3-Dioxygenase; Catechols; Comamonas; DNA, Bacterial; Evolution, Molecular; Fatty Acids, Unsaturated; Hydrolases; Molecular Sequence Data; Multigene Family; Nitrobenzenes; Oxidoreductases; Sequence Analysis, DNA

Oxygen, an essential molecule for life, is utilized not only for cellular respiration but also for biosynthesis and metabolism of various important biomolecules such as steroids, eicosanoids, and neuroactive substances. Since the oxygenases, oxygen-fixating enzymes, were found in 1950s, only stable isotopes (17O and 18O) have been utilized as a tracer for demonstration of oxygen incorporation into organic substances. This stable isotopic method is established, but is hardly applicable to complicated (crude and multi-cellular) systems. Therefore, we here developed a novel radiotracer technique for oxygen metabolism that employs the positron emitter 15O2, whose physical half-life is 2.07 min. In a model reaction with metapyrocatechase, one of the well-known dioxygenases, the substrate catechol was converted to the radioactive product which was identified as alpha-hydroxymuconic epsilon-semialdehyde by a very sensitive LC-radio-UV-MS combined method.

Topics: Catechol 2,3-Dioxygenase; Catechols; Dioxygenases; Electrons; Fatty Acids, Unsaturated; Mass Spectrometry; Oxygen; Oxygen Radioisotopes; Oxygenases; Radioactive Tracers

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.

Topics: Biphenyl Compounds; Catechols; Fatty Acids, Unsaturated; Hydroxybenzoates; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mixed Function Oxygenases; Molecular Structure; Phenols; Pseudomonas; Pyrogallol; Salicylates; Spectrophotometry, Ultraviolet; Substrate Specificity

The xylE gene encodes catechol 2,3-dioxygenase, which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. The expression of this gene in eucaryotic cells can be detected simply by addition of catechol to the growth medium of the cells: cells that have a sufficient level of expression of the xylE gene stain yellow because of the accumulation of 2-hydroxymuconic semialdehyde. The number of stained cells is thus dependent upon the transfection efficiency as well as the level of expression of the xylE gene and is a measure of the combined transfection/expression efficiency in a particular cell type. Since the staining procedure does not affect the viability of the culture, the cells can be harvested afterward and analyzed for the expression of other, cotransfected, genes. This system for measuring transfection efficiency is especially useful when only small amounts of tissue are available.

Topics: Animals; Catechol 2,3-Dioxygenase; Catechols; Chick Embryo; Color; Dioxygenases; Fatty Acids, Unsaturated; Oxygenases; Plasmids; Staining and Labeling; Transfection

1. Two strains of Pseudomonas were grown with phenol and used to prepare cell extracts that metabolized catechol with the transient formation of 2-hydroxymuconic semialdehyde. 2. One of these preparations catalysed the conversion of 1mol. of catechol into 1mol. each of formate and 4-hydroxy-2-oxovalerate. 3. A method for the determination of 4-hydroxy-2-oxovalerate is described, together with some properties of this compound and its 2,4-dinitrophenylhydrazone. 4. Another partially purified cell extract converted 1mol. of 4-hydroxy-2-oxovalerate, formed enzymically from catechol, into 1mol. each of acetaldehyde and pyruvate. This aldolase had a pH optimum of about 8.8, was stimulated by Mg(2+) ions and appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxovalerate.

Topics: Acetaldehyde; Catechols; Chromatography; Fatty Acids; Fatty Acids, Unsaturated; Formates; Fructose-Bisphosphate Aldolase; Metabolism; Phenols; Pseudomonas; Pyruvates; Pyruvic Acid; Research; Valerates