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.

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

“…Chlorocatechol degradation in strain MT1 was thus assumed to occur by a pathway consisting of a patchwork of reactions known from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase), the chlorocatechol pathway (maleylacetate reductase), and a trans-dienelactone hydrolase. However, the kinetic parameters of the characterized muconate cycloisomerase (that is, its preference for 3-chloromuconate over muconate as a substrate) set it apart from previously characterized muconate cycloisomerases (32,53,58). Moreover, salicylate degradation via catechol is usually performed via meta cleavage, and an operon structure comprising genes encoding a LysR type regulator (NahR), a salicylate 1-hydroxylase (NahG), a catechol 2,3-dioxygenase (NahH), and subsequent enzymes of the meta cleavage pathway is usually conserved (2,9,63).…”

“…More importantly, muconate and chloromuconate cycloisomerases differ not only in substrate specificity but also in the reaction performed. Chloromuconate cycloisomerases catalyze a dehalogenation of 3-chloromuconate to form cis-dienelactone (53), and muconate cycloisomerases catalyze the formation of protoanemonin (1) with 4-chloromuconolactone as a reaction intermediate (1,32). 2-Chloromuconate is dehalogenated only by chloromuconate cycloisomerases (59), while mu-conate cycloisomerases form 2-chloro-and 5-chloromuconolactone (60).…”

“…strain MT1 has been reported to degrade 4-and 5-chlorosalicylate by a different metabolic route (32). The substrates are transformed by a salicylate 1-hydroxylase into 4-chlorocatechol, which is subject to ring cleavage by catechol 1,2-dioxygenase to yield 3-chloromuconate.…”

“…The substrates are transformed by a salicylate 1-hydroxylase into 4-chlorocatechol, which is subject to ring cleavage by catechol 1,2-dioxygenase to yield 3-chloromuconate. It is suggested that trans-dienelactone hydrolase acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation in favor of maleylacetate (32). Maleylacetate is reduced to 3-oxoadipate by maleylacetate reductase.…”

“…Moreover, salicylate degradation via catechol is usually performed via meta cleavage, and an operon structure comprising genes encoding a LysR type regulator (NahR), a salicylate 1-hydroxylase (NahG), a catechol 2,3-dioxygenase (NahH), and subsequent enzymes of the meta cleavage pathway is usually conserved (2,9,63). However, no extradiol dioxygenase activity was observed in either salicylategrown or chlorosalicylate-grown cells of MT1 (32), suggesting that the gene encoding salicylate 1-hydroxylase is not clustered with the meta cleavage pathway.…”