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

ABSTRACT 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, C12OccaA, a novel (chloro)muconate cycloisomerase, MCIccaB, 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 (C12OccaA) and ccaB (MCIccaB), 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 C12OccaA and MCIccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCIccaB and the previously identified C12OsalD, rather than C12OccaA, 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.

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

ABSTRACT 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.

New Bacterial Pathway for 4- and 5-Chlorosalicylate Degradation via 4-Chlorocatechol and Maleylacetate in Pseudomonas sp. Strain MT1

ABSTRACT 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.

Formation of Protoanemonin from 2-Chloro-cis,cis-Muconate by the Combined Action of Muconate Cycloisomerase and Muconolactone Isomerase

ABSTRACT 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.

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

ABSTRACT Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate tocis-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 ofcis-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 ascis,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.

Mutations in catB, the Gene Encoding Muconate Cycloisomerase, Activate Transcription of the Distalben Genes and Contribute to a Complex Regulatory Circuit in Acinetobacter sp. Strain ADP1

ABSTRACT Mutants of the bacterium Acinetobacter sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate andcis,cis-muconate to activate expression of thebenABCDE operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by cat gene-encoded enzymes. In this report, four spontaneous mutants were found to carrycatB mutations that enabled BenM-independent growth on benzoate. catB encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate,cis,cis-muconate, is enzymatically produced from catechol by the catA-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the catB mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomalbenA::lacZ transcriptional fusion were constructed and used to investigate how catB mutations affect growth on benzoate. All of the catB mutations increased cis,cis-muconate-activatedben gene expression in strains lacking BenM. A model is presented in which the catB mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular cis, cis-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate ben gene transcription. CatM normally responds to cis,cis-muconate to activate cat gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown catB mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels ofcis,cis-muconate and increasingcatA expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.

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

ABSTRACT Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by convertingcis,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 putidaPRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition tocis,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. putidamuconate 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.