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 P seudomonas 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-β-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.

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1

ABSTRACT 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, C12OccaA, a novel (chloro)muconate cycloisomerase, MCIccaB, 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 (C12OccaA) and ccaB (MCIccaB), 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 C12OccaA and MCIccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCIccaB and the previously identified C12OsalD, rather than C12OccaA, 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.

Crystallization and preliminary crystallographic analysis of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E: a novel dioxygenase involved in the biodegradation of polychlorinated aromatic compounds

Hydroxyquinol 1,2-dioxygenase (HQ1,2O) from Nocardioides simplex 3E, an enzyme involved in the aerobic biodegradation of a large class of chloroaromatic compounds such as 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T), has been crystallized. HQ1,2O, which specifically catalyzes the intradiol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), an intermediate in the degradation of a variety of aromatic pollutants, to maleylacetate, has been recently purified to homogeneity. The enzyme is an homodimer composed of two identical subunits in a α2-type quaternary structure, has a molecular weight of about 65 kDa and contains a catalytically essential Fe(III) ion. Crystals of HQ1,2O obtained using 2% PEG 400 and 2 M ammonium sulfate at pH 7.5 as precipitants belong to the orthorhombic space group P212121, with unit-cell parameters a = 81.15 (6), b = 86.79 (7), c = 114.93 (8). Assuming one dimer per asymmetric unit, the Vm value is 2.51 Å3 Da−1. A complete native data set to 1.8 Å resolution has been collected on a laboratory source. This is the first intradiol dioxygenase which specifically catalyzes the cleavage of hydroxyquinol to give diffraction-quality crystals.