Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho-and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxycis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.Various bacterial cultures which can use chloroaromatics as the single source of carbon and energy for growth, resulting in the formation of carbon dioxide, chloride, and biomass, have been described. These organisms can be differentiated on the basis of the catabolic pathways dealing with the substituents. Chlorosubstituents can be removed by initial oxygenolytic, reductive, or hydrolytic reactions. Further mineralization can then occur via classical pathways such as the 3-oxoadipate and the meta-cleavage pathways. However, the majority of the organisms able to mineralize chlorinated aromatics do not possess enzyme systems capable of initial dechlorination. They transform chlorinated aromatics to chlorocatechols, which are further metabolized by the enzymes of the modified orthocleavage pathway, and dechlorination occurs after ring cleavage (32).It is generally accepted that degradation of chloroaromatics does not proceed via the meta-cleavage pathway (20,26,29). An explanation for this has been found in the production of an acylchloride from 3-chlorocatechol by the catechol 2,3-dioxygenase of the meta-cleavage pathway, which leads to rapid suicide inactivation of the enzyme (3). Therefore, meta cleavage is considered to be unsuitable for the mineralization of haloaromatics that are degraded via halocatechols.Whereas chlorocatechols are mineralized via ortho-cleavage pathways, methylaromatics are commonly mineralized via meta-cleavage routes. Simultaneous metabolism of chloro-and methylcatechols often creates biochemical anarchy. meta cleavage leads to substrate misrouting in the case of 4-chlorocatechol or formation of a suicide product in the case of 3-chlorocatechol. Formation of dead-end methyllactones ca...

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native M r value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ؎ 10 kDa, indicating a homotetrameric enzyme structure (4 ؋ 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ؎ 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a ␤␣␤-element was detected.

A 3,167-bp PstI fragment of genomic DNA from Pseudomonas sp. strain B13 was cloned and sequenced. The gene clcE consists of 1,059 nucleotides encoding a protein of 352 amino acids with a calculated mass of 37,769 Da which showed maleylacetate reductase activity. The protein had significant sequence similarities with the polypeptides encoded by tcbF of pP51 (59.4% identical positions), tfdF of pJP4 (55.1%), and tftE of Burkholderia cepacia AC1100 (53.1%). The function of TcbF as maleylacetate reductase was established by an enzyme assay.

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