Characterization of muconate and chloromuconate cycloisomerase from Rhodococcus erythropolis 1CP: indications for functionally convergent evolution among bacterial cycloisomerases.

Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were purified from extracts of Rhodococcus erythropolis 1CP cells grown with benzoate or 4-chlorophenol, respectively. Both enzymes discriminated between the two possible directions of 2-chloro-cis, cis-muconate cycloisomerization and converted this substrate to 5-chloromuconolactone as the only product. In contrast to chloromuconate cycloisomerases of gram-negative bacteria, the corresponding R. erythropolis enzyme is unable to catalyze elimination of chloride from (+)-5-chloromuconolactone. Moreover, in being unable to convert (+)-2-chloromuconolactone, the two cycloisomerases of R. erythropolis 1CP differ significantly from the known muconate and chloromuconate cycloisomerases of gram-negative strains. The catalytic properties indicate that efficient cycloisomerization of 3-chloro- and 2,4-dichloro-cis,cis-muconate might have evolved independently among gram-positive and gram-negative bacteria.     

Evolutionary Relationship between Chlorocatechol Catabolic Enzymes from Rhodococcus opacus 1CP and Their Counterparts in Proteobacteria: Sequence Divergence and Functional Convergence

Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria. To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified. The clcB gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR. Downstream of clcR but separated from it by 222 bp, we detected the clcA and clcD genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase. A gene coding for a maleylacetate reductase could not be detected. Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS1031 and Tn4811. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of R. opacus 1CP represent different branches in the dendrograms than their proteobacterial counterparts. Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.     

Abundant expression of Pseudomonas genes for chlorocatechol metabolism.

The respective specific activities of catechol 1,2-oxygenase II (catechol 1,2-dioxygenase; EC 1.13.11.1) and muconate cycloisomerase II (chloromuconate cycloisomerase; EC 5.5.1.7) in crude extracts of chlorobenzoate-grown Pseudomonas cells corresponded to about 16 and 11% of the soluble cell protein. High levels of protein synthesis appeared to compensate for a loss in catalytic activity that accompanied evolutionary acquisition of broad substrate specificity required for the enzymes to accommodate halogenated substrates.     

Specificity of catechol ortho-cleavage during para-toluate degradation by Rhodococcus opacus 1cp

Degradation of para-toluate by Rhodococcus opacus 1cp was investigated. Activities of the key enzymes of this process, catechol 1,2-dioxygenase and muconate cycloisomerase, are detected in this microorganism. Growth on p-toluate was accompanied by induction of two catechol 1,2-dioxygenases. The substrate specificity and physicochemical properties of one enzyme are identical to those of chlorocatechol 1,2-dioxygenase; induction of the latter enzyme was observed during R. opacus 1cp growth on 4-chlorophenol. The other enzyme isolated from the biomass grown on p-toluate exhibited lower rate of chlorinated substrate cleavage compared to the catechol substrate. However, this enzyme is not identical to the catechol 1,2-dioxygenase cloned in this strain within the benzoate catabolism operon. This supports the hypothesis on the existence of multiple forms of dioxygenases as adaptive reactions of microorganisms in response to environmental stress.     

Evolution of chlorocatechol catabolic pathways

The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates. They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modifiedortho-cleavage pathway. Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity. In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone. While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant. They seem to share an / hydrolase fold, but the sequences comprising the fold are quite dissimilar. Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway. Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains ofAlcaligenes andBurkholderia (Pseudomonas) cepacia is investigated). Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor. The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.     

Chloromethylmuconolactones as critical metabolites in the degradation of chloromethylcatechols: recalcitrance of 2-chlorotoluene

To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.     

Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate.

The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro- and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Alcaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans-4-carboxymethylene-but-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbon-chlorine bond during their divergence from normal muconate cycloisomerases.     

Enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4-fluorobenzoate.

Enzymatic conversion of 4-fluorocatechol in the simultaneous presence of partially purified preparations of catechol 1,2-dioxygenase from Pseudomonas cepacia and muconate cycloisomerase from Alcaligenes eutrophus 335 yielded a product that was unambiguously identified as (+)-4-fluoromuconolactone [(+)-4-carboxymethyl-4-fluoro-but-2-en-4-olide]. This compound was shown to be the only major product formed from 3-fluoro-cis,cis-muconate by the action of muconate cycloisomerases from A. eutrophus 335, A. eutrophus JMP134, and P. cepacia as well as by the action of dichloromuconate cycloisomerase from A. eutrophus JMP134. This finding implies that dichloromuconate cycloisomerase, like the muconate cycloisomerases, catalyzes primarily a cycloisomerization reaction, which only in the case of chloro- and bromo-substituted substrates is connected to a dehalogenation. 4-Fluoromuconolactone at pH 7 decomposes by spontaneous reactions mainly to maleylacetate, which then decarboxylates to give cis-acetylacrylate. Although significant amounts of an unidentified compound are also formed from the fluorolactone, HF elimination to the two isomeric dienelactones (4-carboxymethylenebut-2-en-4-olides) is negligible. However, all spontaneous reactions proceed so slowly that an enzymatic conversion of 4-fluoromuconolactone must be assumed. Participation of dienelactone hydrolases in this reaction is indicated by their induction during growth of various strains with 4-fluorobenzoate. However, experiments with cell extracts of P. putida A3.12 suggest that at least one other hydrolytic enzyme is able to contribute to 4-fluoromuconolactone conversion. In light of these observations, earlier proposals for a 4-fluorobenzoate degradative pathway are discussed.     

A new type of muconate cycloisomerase from Rhodococcus rhodochrous strain 89

Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloromuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloromuconate cycloisomerases from gram-negative and gram-positive bacteria.     

New bacterial pathway for 4- and 5-chlorosalicylate degradation via 4-chlorocatechol and maleylacetate in Pseudomonas sp. strain MT1

Pseudomonas sp. strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway. 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases. However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone. Protoanemonin is obviously a dead-end product of the pathway. A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates. Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in. As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation. The maleylacetate formed in this way is reduced by maleylacetate reductase. Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH.     

Phenol degradation by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> strain 1G

During cultivation in a liquid medium, the bacterium Rhodococcus opacus 1G was capable of growing on phenol at a concentration of up to 0.75 g/l. Immobilization of Rhodococcus opacus 1G had a positive effect on cell growth in the presence of phenol at high concentrations. The substrate at concentrations of 1.0 and 1.5 g/l was completely utilized over 24 and 48 h, respectively. The key enzymes of phenol degradation (two catechol 1,2-dioxygenases and muconate cycloisomerase) were isolated. One of the dioxygenases was very unstable. By substrate specificity, another enzyme belonged to catechol 1,2-dioxygenases of the classical ortho-pathway. Chlorocatechols and chlorophenols served as competitive inhibitors of catechol 1,2-dioxygenases. The inhibitory effect of other aromatic compounds was less significant. Our results suggest that this strain holds promise for bioremediation of phenol wastewater.     

Enzymology of the beta-ketoadipate pathway in Trichosporon cutaneum.

Cell extracts were prepared from Trichosporon cutaneum grown with phenol or p-cresol, and activities were assayed for enzymes catalyzing conversion of these two carbon sources into 3-ketoadipate (beta-ketoadipate) and 3-keto-4-methyladipate, respectively. When activities of each enzyme were expressed as a ratio, the rate for methyl-substituted substrate being divided by that for the unsubstituted substrate, it was apparent that p-cresol-grown cells elaborated pairs of enzymes for hydroxylation, dioxygenation, and delactonization. One enzyme of each pair was more active against its methyl-substituted substrate, and the other was more active against its unsubstituted substrate. Column chromatography was used to separate two hydroxylase activities and also 1,2-dioxygenase activities; the catechol 1,2-dioxygenases were further purified to electrophoretic homogeneity. Extracts of phenol-grown cells contained only those enzymes in this group that were more active against unsubstituted substrates. In contrast, whether cells were grown with phenol or p-cresol, only one muconate cycloisomerase (lactonizing enzyme) was elaborated which was more active against 3-methyl-cis,cis-muconate than against cis,cis-muconate; in this respect it differed from a cycloisomerase of another strain of T. cutaneum which has been characterized. The cycloisomerase was purified from both phenol-grown and p-cresol-grown cells, and some characteristics were determined.     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C. 1.13.11.1) from Alcaligenes eutrophus CH34

Alcaligenes eutrophus CH34 used benzoate as a sole source of carbon and energy, degrading it through the 3-oxoadipate pathway. All the enzymes required for this degradation were shown to be encoded by chromosomal genes. Catechol 1,2-dioxygenase activity was induced by benzoate, catechol, 4-chlorocatechol, and muconate. The enzyme is most likely a homodimer, with an apparent molecular weight of 76,000 ± 500. According to several criteria, its properties are intermediate between those of catechol 1,2-dioxygenases (CatA) and chlorocatechol 1,2-dioxygenases (ClcA). The determined K _m for catechol is the lowest among known catechol and chlorocatechol dioxygenases. Similar K _m values were found for para-substituted catechols, although the catalytic constants were much lower. The catechol 1,2-dioxygenase from strain CH34 is unique in its property to transform tetrachlorocatechol; however, excess substrate led to a marked reversible inhibition. Some meta- and multi-substituted catechols behaved similarly. The determined K _m (or K _i) values for para- or meta-substituted catechols suggest that the presence of an electron-withdrawing substituent at one of these positions results in a higher affinity of the enzyme for the ligand. Results of studies of recognition by the enzyme of various nonmetabolised aromatic compounds are also discussed. Received: 20 November 1996 / Accepted: 11 April 1996     

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.     

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1

Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12O_ccaA, a novel (chloro)muconate cycloisomerase, MCI_ccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12O_ccaA) and ccaB (MCI_ccaB), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12O_ccaA and MCI_ccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCI_ccaB and the previously identified C12O_salD, rather than C12O_ccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.The aerobic degradation of chloroaromatic compounds usually proceeds via chlorocatechols as central intermediates (20, 47), which in most of the cases reported thus far, are further degraded by enzymes of the chlorocatechol pathway (44). This pathway involves ortho-cleavage by a chlorocatechol 1,2-dioxygenase with high activity for chlorocatechols (12), a chloromuconate cycloisomerase with high activity for chloromuconates (54), a dienelactone hydrolase active with both cis- and trans-dienelactone (4-carboxymethylenebut-2-en-4-olide) (54), and a maleylacetate reductase (MAR) (28).However, it has become evident in recent years that microorganisms have evolved various alternative strategies to mineralize chlorocatechols. Pseudomonas putida GJ31 was found to degrade chlorobenzene rapidly via 3-chlorocatechol using a catechol meta-cleavage pathway (33). Two alternative pathways for 3- and 4-chlorocatechol degradation that involve reactions known from the chlorocatechol, as well as the 3-oxoadipate, pathway have recently been observed in Rhodococcus opacus 1CP (35) and Pseudomonas reinekei MT1 (39). In R. opacus 1CP, 3-chloro- and 2,4-dichloro-cis,cis-muconate (the ring cleavage products of 4-chlorocatechol and 3,5-dichlorocatechol, respectively) are converted to the respective cis-dienelactones (35, 58), similar to the reaction described for proteobacterial chloromuconate cycloisomerases (54). However, proteobacterial chloromuconate cycloisomerase can dehalogenate 2-chloromuconate (the ring cleavage product of 3-chlorocatechol) and transform this compound via 5-chloromuconolactone into trans-dienelactone (54, 65), whereas none of the described chloromuconate cycloisomerases of R. opacus 1CP can catalyze such a dehalogenation, and 5-chloromuconolactone is the product of the cycloisomerization reaction (35, 58). Dehalogenation is achieved by an enzyme with high sequence similarity to muconolactone isomerases (35), which in proteobacteria have been shown to be capable of dehalogenating 5-chloromuconolactone to cis-dienelactone (46).In P. reinekei MT1, a trans-dienelactone hydrolase (trans-DLH) was identified as the key enzyme involved in the degradation of 4- and 5-chlorosalicylate via 4-chlorocatechol as an intermediate (39). In contrast to all previously described dienelactone hydrolases involved in chlorocatechol degradation, which belong to the α/β hydrolase fold enzymes with a catalytic triad consisting of Cys, His, and Asp (10), trans-DLH was shown to be a zinc-dependent hydrolase (8). The function of this enzyme in the 4-chlorocatechol metabolic pathway was to interact with the muconate cycloisomerase (MCI)-mediated transformation of 3-chloromuconate into protoanemonin. By acting on the reaction intermediate 4-chloromuconolactone, trans-DLH prevents the formation of protoanemonin by catalyzing its hydrolysis to maleylacetate (39). Maleylacetate, in turn, is reduced by MAR to 3-oxoadipate.A more detailed genetic and biochemical analysis of the degradation of differently substituted salicylates (7) had shown the presence of two catabolic gene clusters in MT1. An archetype catRBCA gene cluster was shown to be involved in salicylate degradation. The second gene cluster (sal) had a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, clustered with the salCD genes, encoding MCI and catechol 1,2-dioxygenase (C12O), respectively. As these genes were expressed during growth on differently substituted salicylates, it was proposed that the function of the sal gene cluster is to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho-cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways. However, previous analyses had indicated the presence of an additional and thus third (chloro)muconate cycloisomerase in MT1 during growth on chlorosalicylate, which is distinct from both previously described MCIs encoded by the cat cluster (MCI_catB) and the sal cluster (MCI_salC), as it transforms 3-chloromuconate into approximately equal amounts of cis-dienelactone and protoanemonin (39). In the present report, this cycloisomerase is biochemically and genetically described and shown to be located in a third gene cluster involved in the degradation of 5-chlorosalicylate by strain MT1. This cluster comprises genes encoding a third C12O, trans-DLH (8), and a MAR. Evidently, P. reinekei MT1 is the first microorganism in which such a complex net of genes involved in chlorocatechol degradation has been described.     

Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-cis,cis-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A

Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate to cis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation of cis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such as cis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate. Interestingly, cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.