The meta Cleavage of Catechol by Azotobacter Species

Abstract

1. Catechol was metabolized through 2-hydroxymuconic semialdehyde by cell-free extracts of benzoate-grown Azotobacter strains. Some properties of catechol 2,3 oxygenase preparations from Azotobacter vinelandii 206 are described.2 . Two different enzymatic activities able to attack 2-hydroxymuconic semialdehyde have been found in crude extracts from benzoate-grown cells ; one catalyses a hydrolytic release of formate from the semialdehyde and the other a dehydrogenation of this compound to 4-oxalocrotonate. However, the low, non-inducible levels of 2-hydroxymuconic semialdehyde hydrolase activity appear negligible for metabolic purposes and the semialdehyde seems to be dissimilated almost exclusively via 4-oxalocrotonate, by the action of a NAD+-dependent dehydrogenase, in Azotobacter strains.3, A tautomerase activity responsible for the interconversion of the enol and keto forms of 4-oxalocrotonic acid was found in extracts from benzoate-grown cells.4. 4-Oxalocrotonate was stoicheiometrically converted to CO, and 4-hydroxy-2-oxovalerate by a partially purified extract, with the transient formation of a compound that appears to be 2-oxopent-4-enoic acid. The 4-oxalocrotonate decarboxylase activity was stimulated by Mg2+ or Mn2+ ions and was inhibited by EDTA.5. Cell-free extracts from Azotobacter strains converted synthetic 4-hydroxy-2-oxovalerate to acetaldehyde and pyruvate.6. A reaction sequence, termed the 4-oxalocrotonate pathway, for the dissimilation of catechol to acetaldehyde and pyruvate by Azotobacter species is presented. All the enzymes operative in this pathway were inducible, except the 4-hydroxy-2-oxovalerate aldolase. 7. The findings described here are discussed in connection with the two previously reported meta cleavage pathways for the oxidation of catechol in Pseudomonas strains.

Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture

“…Extracts of culture cells sampled from the chemostats were prepared by sonication and centrifugation. Subsequently, catechol 2,3-dioxygenase activity was determined according to the method of Sala-Trepat & Evans (1971).…”

The Metabolism of Benzoate and Methylbenzoates via the meta‐Cleavage Pathway by Pseudomonas arvilla mt‐2

“…The procedures for assaying and definition of the units of activity of catechol2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, 2-hydroxymuconic semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase and 4-hydroxy-2-oxovalerate aldolase were as previously described [3,8].…”

“…Sala-Trepat and Evans [3] elucidated the two resulting pathways, which after diverging a t 2-hydroxymuconic semialdehyde converge again a t 2-oxopent-4-enoate ; they concluded that in Azotobacter only the 4-oxalocrotonate branch, involving as the first step the NADf-dependent dehydrogenation of 2-hydroxymuconic semialdehyde, was of any metabolic significance since the 2-hydroxymuconic semialdehyde hydrolase was present in only very low uninducible levels. I n NCIB 9816 all the enzymes of the 4-oxalocrotonate branch are present [4] and again it has been proposed that catechol is metabolised mainly by this pathway, since although the hydrolase is induced by growth on naphthalene, the level of induction is much less than that of the 2-hydroxymuconic semialdehyde dehydrogenase.…”

End-product control of expression of branched-chain amino acid biosynthesis genes in Streptomyces coelicolor A3(2): paradoxical relationships between DNA sequence and regulatory phenotype The GenBank accession numbers for the ilvD and leuA sequences determined in this work are AF068843 and AF026444, respectively.

“…Enzyme and protein assays. CO was assayed by the spectrophotometric method of Sala-Trepat & Evans (1971) with the following modifications : the increase in absorbance at 375 nm was continuously monitored with the cuvette maintained at 30 mC ; the final volume was 1 ml ; 100 µmol assay phosphate buffer (100 mM NaH # PO % , 100 mM K # HPO % , pH 8n0) and 100 µl enzyme extract were used. Control checks for reproducibility were performed by measuring the enzyme activity of the extract obtained from unsupplemented biomass after storage on ice for 3 h ; the variations were slight (2 % or less) and did not account for the between-experiment variability commented on in Results.…”