Degradation of 2,3-dihydroxybenzoate by a novel meta-cleavage pathway

Abstract

2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via meta-cleavage of the aromatic ring. The dhb cluster of Pseudomonas reinekei MT1 encodes a chimeric meta-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-p-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of P. reinekei MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the dhB gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-p-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate.

Fig 1

Chimeric organization of the dhb gene cluster. Organization and comparison of the dhb gene cluster of P. reinekei MT1 to the cmt cluster of P. putida F1 and the phenol catabolic gene cluster comprising meta-cleavage pathway genes of C. necator H16. Genes with high similarity to those of the dhb gene cluster are framed in boldface. HMS, 2-hydroxymuconic semialdehyde; HCOMODA, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate.

Fig 2

Dendrograms showing the relatedness of Dhb enzymes. (A) Extradiol dioxygenases; (B) decarboxylases of the class II aldolase family acting in aromatic metabolism; (C) 2-oxopent-4-enoate hydratase and 4-oxocrotonate decarboxylase; (D) acetaldehyde dehydrogenase; and (E) 2-hydroxymuconic semialdehyde hydrolases. The evolutionary history was inferred using the neighbor-joining method and the p-distance model after alignment of sequences using MUSCLE (19). All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons. Phylogenetic analyses were conducted in MEGA5 (53).

Fig 3

Proposed pathway for 2,3-DHB degradation by P. reinekei MT1. A putative route for 2,3-dihydroxy-p-cumate metabolism is also shown. Enzymes used include the following: DhbA, 2,3-DHB 3,4-dioxygenase; DhbB, 2-hydroxy-3-carboxymuconic semialdehyde decarboxylase; DhbC, 2-hydroxymuconic semialdehyde dehydrogenase; DhbD, 2-oxopent-4-enoate hydratase; DhbE, 4-oxalocrotonate decarboxylase; DhbF, 4-oxalocrotonate isomerase; DhbG, acetaldehyde dehydrogenase; DhbH, 4-hydroxy-2-oxovalerate aldolase; and DhbI, 2-hydroxymuconic semialdehyde hydrolase.

Fig 4

Conversion of 2,3-DHB by extracts of E. coli JM109(pGC23O) and structure of the ring cleavage product as deduced by ¹H NMR analysis. The sample for photometric analysis contained 50 mM phosphate buffer (pH 8.0) and 50 μM 2,3-DHB. Spectra were recorded before the addition of 15 μl of cell extract for 20 min at 2-min intervals. ¹H NMR analysis was performed after transformation of 1 mM 2,3-DHB in 50 mM phosphate buffer (pH 8.0).

Fig 5

Absolute (top) and relative (bottom) expression levels of catabolic genes in 2,3-DHB-grown cells of P. reinekei MT1 as determined by quantitative real-time PCR. The number of transcripts/ng of cDNA in gluconate (light gray bars) and 2,3-DHB-grown cells (dark gray bars) is shown (top). The error bars indicate standard deviations. Relative expression values (bottom) represent n-fold changes in the ratio of gene expression between the target gene and the reference gene (rpsL) compared to expression under noninducing conditions (for rpsL, this ratio was set as 1).