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

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.

Crystal Structure and Catalytic Mechanism of 4-Methylmuconolactone Methylisomerase

“…The encoding gene has been localized in the genome of C. necator JMP134 (GenBank TM accession numbers: AAZ60870 and CAA67958) (8), and a putative MLMI encoding gene has been localized on plasmid pHG1 of C. necator H16 (GenBank TM accession number: AAP86134). P. reinekei MT1 also contains MLMI-like activity for the isomerization of 4-ML, as recently shown by Camara et al (1).…”

“….0]octane (1-methylbislactone), and 3-methyl-cis,cis-muconate were prepared as previously described (1,4,6).…”

“…ria degrade methyl-substituted aromatics via meta (extradiol)-cleavage of methylcatechols, this strain specializes in the degradation of such compounds via ortho (intradiol)-cleavage (1). Ortho-cleavage of 4-methylcatechol typically results in the formation of 4-methylmuconolactone (4-ML) 2 as a dead-end product (1,3,4).…”

“…Ortho-cleavage of 4-methylcatechol typically results in the formation of 4-methylmuconolactone (4-ML) 2 as a dead-end product (1,3,4).…”

Degradation of 2,3-Dihydroxybenzoate by a Novel meta -Cleavage Pathway

“…The dhb gene cluster was localized by sequencing downstream of the previously described sal cluster of P. reinekei MT1, which is harbored on a fosmid from a previously constructed fosmid library (9). Direct sequencing was performed using the ABI Prism BigDye Terminator v1.1 ready reaction cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).…”