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.

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.

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.A major route for mineralization of chloroaromatic compounds by microorganisms is their transformation into chlorocatechols and their further metabolism by enzymes of the chlorocatechol pathway (24). In this metabolic pathway, chlorocatechols are subject to intradiol cleavage to form the respective chloromuconates, which are converted by chloromuconate cycloisomerases into cis-or trans-dienelactone. The dienelactones undergo hydrolysis by dienelactone hydrolase. The hydrolysis product formed during the metabolism of 4-chlorocatechol was tentatively identified as "maleylacetate" based on its absorption characteristics ( max ϭ 243 nm in aqueous alkali) by Evans et al. (11). Similarly, Tiedje et al. (31) postulated maleylacetate and chloromaleylacetate as products formed during the metabolism of 4-chloro-and 3,5-dichlorocatechol, respectively. Those authors noted, however, that UV absorption was essentially quenched upon acidification, a behavior resembling keto-enol tautomerism, raising the question of the actual solution structure of maleylacetate. More recently, Seibert et al. claimed that the enol form (3-hydroxy-2,4-hexadienedioate, 3-hydroxymuconate) is thermodynamically favored under physiological conditions and exhibits an absorption maximum at 243 nm (28). The disappearance of this absorption under acidic conditions was believed to be due to the presence of the keto form, 3-oxo-cis-4-hexenedioate (maleylacetate in the strict sense), under these conditions. Despite the authors' assumption that 3-hydroxymuconate was the actual substrate of the purified reductase, the enzyme was termed "maleylacetate" reductase. Later, Prucha et al. (23) showed that the hydrolysis product of 3-methyldienelactone, supposedly 3-methylmaleylacetate or 3-hydroxy-4-methylmuconate, has a cyclic structure under acidic conditions (4-carboxymethylene-4-hydroxy-3-methylbut-2-en-4-olide, 4-hydroxy-3-methylmuconolactone), while no indication of the configuration under physiological conditions was given. Thus, although the structure of the decarboxylation product has been published (27), the actual solution structure of "maleylacetate" remained uncertain, presumably due to its reportedly high instability."Maleylacetate" is an intermediate not only in the degradation of chlo...

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.