Modified 3-oxoadipate pathway for the biodegradation of methylaromatics in Pseudomonas reinekei MT1

Abstract

Catechols are central intermediates in the metabolism of aromatic compounds. Degradation of 4-methylcatechol via intradiol cleavage usually leads to the formation of 4-methylmuconolactone (4-ML) as a dead-end metabolite. Only a few microorganisms are known to mineralize 4-ML. The mml gene cluster of Pseudomonas reinekei MT1, which encodes enzymes involved in the metabolism of 4-ML, is shown here to encode 10 genes found in a 9.4-kb chromosomal region. Reverse transcription assays revealed that these genes form a single operon, where their expression is controlled by two promoters. Promoter fusion assays identified 4-methyl-3-oxoadipate as an inducer. Mineralization of 4-ML is initiated by the 4-methylmuconolactone methylisomerase encoded by mmlI. This reaction produces 3-ML and is followed by a rearrangement of the double bond catalyzed by the methylmuconolactone isomerase encoded by mmlJ. Deletion of mmlL, encoding a protein of the metallo-beta-lactamase superfamily, resulted in a loss of the capability of the strain MT1 to open the lactone ring, suggesting its function as a 4-methyl-3-oxoadipate enol-lactone hydrolase. Further metabolism can be assumed to occur by analogy with reactions known from the 3-oxoadipate pathway. mmlF and mmlG probably encode a 4-methyl-3-oxoadipyl-coenzyme A (CoA) transferase, and the mmlC gene product functions as a thiolase, transforming 4-methyl-3-oxoadipyl-CoA into methylsuccinyl-CoA and acetyl-CoA, as indicated by the accumulation of 4-methyl-3-oxoadipate in the respective deletion mutant. Accumulation of methylsuccinate by an mmlK deletion mutant indicates that the encoded acetyl-CoA hydrolase/transferase is crucial for channeling methylsuccinate into the central metabolism.

FIG. 1.

Present status of knowledge on ortho cleavage pathway for catechol (top) or 4-methylcatechol (bottom) degradation. Metabolic routes for 4-methylcatechol have been proposed for C. necator JMP134 (lower branch) and R. rhodochrous N75 (upper branch). Enzyme names are as follows: 1, catechol 1,2-dioxygenase; 2, muconate cycloisomerase; 3, 4-methylmuconolactone methylisomerase; 4, muconolactone or methylmuconolactone isomerase; 5, 3-oxoadipate enol-lactone hydrolase; 6, 3-oxoadipate:succinyl-CoA transferase; 7, 3-oxoadipyl-CoA thiolase; 8, 3-methylmuconolactone-CoA synthetase; ?, unknown enzymes.

FIG. 2.

Comparison of the mml gene cluster of P. reinekei MT1 with those of C. necator JMP134 and C. necator H16. Genes encoding catabolic enzymes, transcriptional regulators, and putative transporters are indicated in light gray, gray, and dark gray, respectively. Genes framing the mml gene clusters are indicated in black. Genes essential for growth on 4-methylsalicylate are indicated with black triangles, whereas those that are dispensable are indicated with unfilled triangles. Numbers below the arrows indicate the primer pairs utilized to assess transcription of intergenic regions. Double-headed arrows represent intergenic regions transcribed during growth on 4-methylsalicylate (MT1) or 4-methylbenzoate (JMP134::X), and lines indicate those regions not transcribed. Experimentally determined promoter regions are indicated by curved arrows.

FIG. 3.

Proposed pathway for 4-ML degradation by P. reinekei MT1. Metabolites identified in the current study are depicted in boxes. MmlI, 4-methylmuconolactone methylisomerase; MmlJ, methylmuconolactone isomerase; MmlL, 4-methyl-3-oxoadipate enol-lactone hydrolase; MmlFG, 4-methyl-3-oxoadipate-CoA transferase; MmlC, 4-methyl-3-oxoadipyl-CoA thiolase; MmlK, acetyl-CoA-transferase/hydrolase.