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.

Metabolic functions of Pseudomonas fluorescens strains from Populus deltoides depend on rhizosphere or endosphere isolation compartment

“…Homologs of the cmtC and cmtD genes from the p -cumate degradation pathway were identified, which encode for 2,3-dihydroxy- p -cumate dioxygenase and a decarboxylase, followed by a set of genes whose products feed the resulting degradation intermediate into the meta -cleavage pathway (Eaton, 1996 ) in 3/4 rhizosphere and 5/15 endosphere isolates. These genes are likely involved in degradation of the plant metabolite 2,3-dihydroxybenzoate as observed in P. fluorescens (DeFrank and Ribbons, 1977 ) and P. reinekei MT1 (Marín et al, 2012 ).…”

2,3-Dihydroxybenzoate meta-Cleavage Pathway is Involved in o-Phthalate Utilization in Pseudomonas sp. strain PTH10

“…To identify the OPA degradation genes in strain PTH10, we first attempted to isolate the 23DHBA 3,4-dioxygenase gene. The 23DHBA meta -cleavage pathway in Pseudomonas reinekei MT1 has been reported by Marín and coworkers 22 . The extradiol ring cleavage dioxygenase, 23DHBA 3,4-dioxygenase (DhbA; accession number, AFN52421), is responsible for ring cleavage of 23DHBA in this species.…”

“…When 100 μM 23DHBA, which has maximum absorption at 240 nm, was incubated with the cell extract containing OpaC at 30 °C, it was converted to a product with maximum absorption at 343 nm (see Fig. S1a in the supplemental material), which is identical to that of CHMS 22 . No conversion was observed when the cell extract of E. coli harboring the empty vector was used (data not shown), indicating that 23DHBA was converted to CHMS by the opaC -encoding 23DHBA 3,4-dioxygenase.…”

“…Degradation via 23DHBA is notably different from other known OPA catabolic pathways 12,14 . Although, the 23DHBA meta -cleavage pathway has been found in P. reinekei MT1 22 , growth ability on OPA and the genes responsible for its conversion to 23DHBA have never been reported. The amino acid sequence of OpaC presents similarity to that of DhbA from MT1, which belongs to the type I extradiol dioxygenase family 26 .…”

“…In the presence of either OpaC or DhbA, CHMS was generated as a reaction product from 23DHBA, suggesting that these enzymes share a similar reaction mechanism for the aromatic ring cleavage of 23DHBA. The dhbB gene-encoding class II aldolase is involved in the decarboxylation of CHMS in MT1 22 . In contrast, the CHMS decarboxylation of strain PTH10 requires OpaD, which belongs to a metallo-dependent hydrolase superfamily 27 .…”