Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998     

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.     

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.     

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.     

Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.     

Formation of protoanemonin from 2-chloro-cis,cis-muconate by the combined action of muconate cycloisomerase and muconolactone isomerase

Muconate cycloisomerases are known to catalyze the reversible conversion of 2-chloro-cis,cis-muconate by 1,4- and 3,6-cycloisomerization into (4S)-(+)-2-chloro- and (4R/5S)-(+)-5-chloromuconolactone. 2-Chloromuconolactone is transformed by muconolactone isomerase with concomitant dechlorination and decarboxylation into the antibiotic protoanemonin. The low k(cat) for this compound compared to that for 5-chloromuconolactone suggests that protoanemonin formation is of minor importance. However, since 2-chloromuconolactone is the initially predominant product of 2-chloromuconate cycloisomerization, significant amounts of protoanemonin were formed in reaction mixtures containing large amounts of muconolactone isomerase and small amounts of muconate cycloisomerase. Such enzyme ratios resemble those observed in cell extracts of benzoate-grown cells of Ralstonia eutropha JMP134. In contrast, cis-dienelactone was the predominant product formed by enzyme preparations, in which muconolactone isomerase was in vitro rate limiting. In reaction mixtures containing chloromuconate cycloisomerase and muconolactone isomerase, only minute amounts of protoanemonin were detected, indicating that only small amounts of 2-chloromuconolactone were formed by cycloisomerization and that chloromuconate cycloisomerase actually preferentially catalyzes a 3,6-cycloisomerization.     

Structural basis for the substrate specificity and the absence of dehalogenation activity in 2-chloromuconate cycloisomerase from Rhodococcus opacus 1CP

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate. Furthermore, Rho-2-CMCI is not able to dehalogenate the 5-chloromuconolactone and therefore it cannot generate the dienelactone.     

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.     

Detoxification of Protoanemonin by Dienelactone Hydrolase

Protoanemonin is a toxic metabolite which may be formed during the degradation of some chloroaromatic compounds, such as polychlorinated biphenyls, by natural microbial consortia. We show here that protoanemonin can be transformed by dienelactone hydrolase of Pseudomonas sp. strain B13 to cis-acetylacrylate. Although similar K_m values were observed for cis-dienelactone and protoanemonin, the turnover rate of protoanemonin was only 1% that of cis-dienelactone. This indicates that at least this percentage of the enzyme is in the active state, even in the absence of activation. The trans-dienelactone hydrolase of Pseudomonas sp. strain RW10 did not detectably transform protoanemonin. Obviously, Pseudomonas sp. strain B13 possesses at least two mechanisms to avoid protoanemonin toxicity, namely a highly active chloromuconate cycloisomerase, which routes most of the 3-chloro-cis,cis-muconate to the cis-dienelactone, thereby largely preventing protoanemonin formation, and dienelactone hydrolase, which detoxifies any small amount of protoanemonin that might nevertheless be formed.     

Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP.

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.     

Catabolism of 3,5-dichlorosalicylate by Pseudomonas species strain JWS

Abstract A Pseudomonas sp. strain JWS was isolated from an enrichment culture with 3,5-dichlorosalicylate as the sole source of carbon and energy. Additionally, 3-chloro-, 5-chloro-, and 3,5-dibromosalicylate, but not 4-chlorosalicylate were mineralized by the organism. During growth on the chlorosalicylates, stoichiometric amounts of chloride were released into the culture medium. In the presence of both salicylate and 3,5-dichlorosalicylate, high activities were induced for the turnover of non-halogenated as well as halogenated salicylates. Enzyme activities assayed in crude cell extracts which are responsible for the oxidation of catechol and its halogenated derivatives as well as those for cycloisomerization of cis,cis -muconate and its 2,4-dichloro derivative provided indications for the involvement of inducible type II catechol 1,2-dioxygenase and muconate cycloisomerase in biodegradation of halogenated salicylates.     

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.     

Molecular properties of cis,cis-muconate cycloisomerase from Pseudomonas putida

cis,cis-Muconate cycloisomerase (cis,cis-muconate lactonizing enzyme, EC 5.5.1.1.) was purified in crystalline form from Pseudomonas putida. Ultracentrifugation studies, as well as gel filtration chromatography and electrophoresis, indicate that the enzyme is an oligomeric protein of molecular weight 252,000 (s_20,w 12.20 × 10^?13 s), which is built of six homologous protomers of molecular weight 42,000. Studies of enzyme crystals and enzyme molecules in the electron microscope suggest that the cis,cis-muconate cycloisomerase is a hexamer in which the six protomers are arranged in a dihedral point-group symmetry 32 (D₃). Each protomer has a diameter of 42.5Åand six protomers are associated in a structure with a trigonal antiprismatic geometry (a hexamer D₃ octahedron). This model could account for the dimensions most frequently observed by negative staining of the enzyme in solution. A model for the three-dimensional structure of enzyme crystals in which each hexameric enzyme molecule is surrounded by eight neighbouring enzyme molecules, is described.     

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis, cis-muconate. cis, cis -muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or β-ketoadipate pathway. The catBC operon encodes two enzymes of the β-ketoadipate pathway and also requires CatR and cis, cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis, cis-muconate, but only forms a single complex (C2) in the presence of cis, cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis, cis -muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis, cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.     

Conversion of 2-Fluoromuconate to cis-Dienelactone by Purified Enzymes of Rhodococcus opacus 1cp

The present study describes the ¹⁹F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.     

Conversion of 2-chloro-cis,cis-muconate and its metabolites 2-chloro- and 5-chloromuconolactone by chloromuconate cycloisomerases of pJP4 and pAC27.

2-Chloro-cis,cis-muconate, the product of ortho-cleavage of 3-chlorocatechol, was converted by purified preparations of the pJP4- and pAC27-encoded chloromuconate cycloisomerases (EC 5.5.1.7) to trans-dienelactone (trans-4-carboxymethylenebut-2-en-4-olide). The same compound was also formed when (+)-2-chloro- and (+)-5-chloromuconolactone were substrates of these enzyme preparations. Thus, the pJP4- and pAC27-encoded chloromuconate cycloisomerases are able to catalyze chloride elimination from (+)-5-chloromuconolactone. The ability to convert (+)-2-chloromuconolactone differentiates these enzymes from other groups of cycloisomerases.     

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.     

Accumulation of 2-chloromuconate during metabolism of 3-chlorobenzoate by Alcaligenes eutrophus JMP134

Alcaligenes eutrophus JMP134 metabolizes 3-chlorobenzoate via 3- (3CC) and 4-chlorocatechol (4CC) as central metabolites. Whereas 4CC was efficiently degraded without a build-up of significant quantities of intermediates, substantial amounts of 2-chloro-cis,cis-muconate (2CM) formed from 3CC were excreted as a result of the poor activity of dichloromuconate cycloisomerase for this compound. This pathway bottleneck can, using appropriate fermentation conditions, be exploited in the production of 2CM. Correspondence to: D. H. Pieper     

Biodegradation of 3-chlorobenzoate by Pseudomonas putida 10.2

Pseudomonas putida 10.2, a 3-chlorobenzoate (3CBa)-degrading bacterium, was isolated from a soil sample obtained from an agricultural area in Chiang Mai, Thailand. This bacterium could degrade 2mm 3CBa very rapidly with the concomitant formation of chloride ion when grown in mineral salt-yeast extract medium. The presence of glucose, lactose and pyruvate in the medium reduced the capability of this bacterium to degrade 3CBa. Metabolites such as 3-chlorocatechol (3CC), catechol and cis,cis-muconic acid (muconate) could be detected in the growth medium or in cell suspensions when 3CBa was used as the substrate. Furthermore, when crude enzyme extract prepared from 3CBa-grown P. putida 10.2 was incubated with 3CC, catechol and muconate could be detected in the reaction mixtures. Thus, the biodegradation pathway of 3CBa by P. putida 10.2 was proposed to involve transformation of 3CBa to 3CC. The dehalogenation step is believed to involve removal of chloride from 3CC to form catechol, which is subsequently converted to muconate.