RESUMO
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.
Assuntos
Proteínas de Bactérias/fisiologia , Catecóis/metabolismo , Liases Intramoleculares/fisiologia , Família Multigênica/genética , Família Multigênica/fisiologia , Pseudomonas/enzimologia , Pseudomonas/metabolismo , Proteínas de Bactérias/genética , Liases Intramoleculares/genética , Cinética , Dados de Sequência Molecular , Filogenia , Pseudomonas/genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
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.
Assuntos
Catecóis/metabolismo , Família Multigênica , Pseudomonas/genética , Salicilatos/metabolismo , Sequência de Bases , Catecol 1,2-Dioxigenase/metabolismo , Liases Intramoleculares/isolamento & purificação , Liases Intramoleculares/metabolismo , Oxigenases de Função Mista/isolamento & purificação , Dados de Sequência Molecular , Pseudomonas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Sórbico/análogos & derivados , Ácido Sórbico/metabolismoRESUMO
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.
Assuntos
Catecóis/metabolismo , Dioxigenases , Maleatos/metabolismo , Complexos Multienzimáticos/metabolismo , Pseudomonas/metabolismo , Salicilatos/metabolismo , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Catecol 1,2-Dioxigenase , Furanos/análise , Furanos/metabolismo , Genoma Bacteriano , Liases Intramoleculares/metabolismo , Maleatos/análise , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Oxigenases/metabolismo , Pseudomonas/genética , Homologia de Sequência de Aminoácidos , Xenobióticos/metabolismoRESUMO
A (1)H nuclear magnetic resonance ((1)H NMR) assay was used to study the enzymatic transformation of cis-dienelactone, a central intermediate in the degradation of chloroaromatics. It was shown that the product of the cis-dienelactone hydrolase reaction is maleylacetate, in which there is no evidence for the formation of 3-hydroxymuconate. Under acidic conditions, the product structure was 4-carboxymethyl-4-hydroxybut-2-en-4-olide. Maleylacetate was transformed by maleylacetate reductase into 3-oxoadipate, a reaction competing with spontaneous decarboxylation into cis-acetylacrylate. One-dimensional (1)H NMR in (1)H(2)O could thus be shown to be an excellent noninvasive tool for monitoring enzyme activities and assessing the solution structure of substrates and products.