The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase. In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWWO of Pseudomonas putida mt-2 were purified and characterized. The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500. The decarboxylase and the hydratase were 27-and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns. The structural genes for the decarboxylase (xyll) and the hydratase (xylj) were cloned into Escherichia coli. The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E. coli XyII+ XylJ. and XyllI XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria. This suggests that the carboxylase and the hydratase form a complex in vivo. The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase. The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate. The hydratase acts on the former but not the latter isomer. Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo.Enzymes encoded by TOL plasmids metabolize toluene and some of its substituted derivatives via meta cleavage of catechol (2, 14; Fig. 1). The pathway diverges into hydrolytic and dehydrogenative routes at the ring fission product (Fig. 1, compound 2) of catechol (compound 1) and reconverges later at 2-hydroxypent-2,4-dienoate (compound 4). The hydrolytic branch converts the ring fission product (compound 2) directly to compound 4 through the action of hydroxymuconic semialdehyde hydrolase, whereas the dehydrogenative branch involves formation of 2-hydroxyhexa-2,4-diene-1,6-dionate (the enol form of 4-oxalocrotonate) or its methyl substituents (compound 3a) by NAD+-dependent hydroxymuconic semialdehyde dehydrogenase, which is then converted to compound 4 by two enzymatic steps catalyzed by 4-oxalocrotonate isomerase and 4-oxalocrotonate decarboxylase (17). Each of these branched pathways metabolizes different compounds at different efficiencies. The ring fission product of 3-methylcatechol, 2-hydroxy-6-oxohepta-2,4-dienoat...

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2" for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobiis Ql. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3-and 3,4-dihydroxybiphenyl, catechol, and 3-methyl-and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalenedegrading bacteria.Amino-and hydroxynaphthalenesulfonic acids (ANS and HNS, respectively) serve as building blocks for the largescale synthesis of azo dyes. Since arylsulfonates are very rare among natural compounds (21), naphthalenesulfonic acids are referred to as xenobiotics. Arylsulfonates are major pollutants of the environment, and the contamination of the Rhine River by sulfur-organic compounds is largely due to this class of compounds (25). Nevertheless, bacteria which degrade naphthalenesulfonic acids have repeatedly been isolated (4, 5, 29-31, 41, 43). Brilon et al. (4, 5) selected pseudomonads which degraded naphthalene-2-sulfonic acid (2NS) and naphthalene-1-sulfonic acid (1NS). The initial reaction in the degradation of iNS and 2NS is catalyzed by a 1,2-dioxygenase which weakens the carbon-sulfur bond.

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The Protocatechuate 3,4-dioxygenase ( Fig. 1; EC 1.13.11.3) plays an essential role in the utilization of numerous aromatic and hydroaromatic compounds via the P-ketoadipate pathway (45). Ubiquity of the pathway in the natural environment is indicated by the fact that the enzyme is a trait universally shared by fluorescent Pseudomonas species (46) and by members of the family Rhizobiaceae (39). The available evidence points to a single evolutionary origin for protocatechuate 3,4-dioxygenases formed by diverse bacteria (14). Other bacterial intradiol dioxygenases (Fig. 1) act upon catechol. Catechol oxygenase I (40) exhibits narrow substrate specificity and a relatively high Kcat (33) when compared with catechol oxygenase II (10), an enzyme that more readily accommodates chlorocatechol as a substrate and exhibits a relatively low Kcat (Fig. 1).The activity of intradiol dioxygenases depends on ferric ion which is ligated by two histidyl and two tyrosyl side chains within the catalytic subunit of the enzymes (41). Determination of the crystal structure of a Pseudomonas protocatechuate 3,4-dioxygenase has established the position of the iron-ligating histidyl and tyrosyl residues within the primary sequence of the a subunit (34). Like other protocatechuate 3,4-dioxygenases, the Pseudomonas enzyme contains in equimolar amounts the i subunit and an a subunit that contributes to substrate binding. Amino acid sequence comparisons (21,24)

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The lB-ketoadipate pathway of Acinetobacter caloaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded by the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); the other branch, catalyzed by products of the pca genes, converts protocatechuate to succinate and acetyl-CoA by six metabolic reactions analogous or identical to those of the catechol sequence. We used the expression plasmid pUC18 to construct expression libraries of DNA from an A. caloaceticus mutant strain from which the cat genes had been deleted. Immunological screening with antiserum to the pcaE gene product, ,B-ketoadipate:succinyl-CoA transferase I, resulted in the isolation of a cloned 11-kilobase-pair (kbp) fragment which inducibly expressed all six pea genes under control of the lac promoter on pUC18. The induced Escherichia coli cells formed the six pea gene products at levels 10-to 30-fold higher than found in fully induced A. calcoaceicus cultures, although protocatechuate 3,4-dioxygenase (the iron-containing product of the pcaA gene) from the recombinant strain possessed a relatively low turnover number. An E. coli culture expressing the cloned pea genes quantitatively converted protocatechuate to (-ketoadipate; failure of the organism to metabolize the latter compound can be most readily ascribed to relatively low pool levels of succinyl-CoA, a required substrate for 0-ketoadipate:succinyl-CoA transferase, in E. coli. The gene order and direction of transcription were determined to be pcACBDFE by identification of enzymes expressed in subclones, by using natural transformation to identify subclones carrying DNA corresponding to dysfunctional alleles in mutant A. calcoaceticus strains, and by restriction mapping of both the ll-kbp fragment and derivatives of the ll-kbp fragment containing TnS in the pcaA, pcaB, peaC, pcaD, and peaE genes. The fragment containing the pea gene hybridized strongly and specifically to a previously cloned fragment containing A. caloaceticus cat genes.The pca structural genes encode six enzymes that convert protocatechuate to citric acid cycle intermediates via Pketoadipate ( Fig. 1; 27). Protocatechuate induces coordinate synthesis of the enzymes in Acinetobacter calcoaceticus (3), and their unified transcriptional control has been suggested by their constitutive formation in regulatory mutants (4,22). The physical organization of the A. calcoaceticus pca genes has not been explored. The A. calcoaceticus cat genes encode six enzymes that convert catechol to citric acid cycle intermediates by reactions analogous or identical to those catalyzed by the pca gene products (Fig. 1) To establish a basis for analysis of possible interaction between cat and pca genes in A. calcoaceticus, we constructed expression libraries of A. calcoaceticus DNA and used immunological screening to identify a clone that expressed the pcaE gene. The cloned DNA was an 11-kbp EcoRI fragment that hybridized with the 5-kbp EcoRI fragment containing the catBCDE genes and pos...