Characterization of the Protocatechuate 4,5-Cleavage Pathway Operon in Comamonas sp. Strain E6 and Discovery of a Novel Pathway Gene

Abstract

The protocatechuate (PCA) 4,5-cleavage (PCA45) pathway is the essential catabolic route for the degradation of various aromatic acids in the genus Comamonas. All of the PCA45 pathway genes, orf1-pmdKEFDABC, as well as another PCA 4,5-dioxygenase gene, pmdA II B II , were isolated from a phthalate-degrading bacterium, Comamonas sp. strain E6. Disruption of pmdB and pmdD in E6, which code for the ␤ subunit of PCA 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate (PDC) hydrolase, respectively, resulted in a growth defect on PCA, indicating that these genes are essential for the growth of E6 on PCA. On the other hand, inactivation of pmdB II did not affect the growth of E6 on PCA. Disruption of pmdK, which is related to a 4-hydroxybenzoate/ PCA transporter of Pseudomonas putida, resulted in growth retardation on PCA. The insertional inactivation of orf1 in E6, whose deduced amino acid sequence has no similarity with proteins of known function, led to the complete loss of growth on PCA and the accumulation of PDC and 4-oxalomesaconate (OMA) from PCA. These results indicated the involvement of orf1 in the PCA45 pathway, and this gene, designated pmdU, was suggested to code for OMA tautomerase. Reverse transcription-PCR analysis suggested that the pmdUKEFDABC genes constitute an operon. The transcription start site of the pmd operon was mapped at 167 nucleotides upstream of the initiation codon of pmdU. The pmd promoter activity was enhanced 20-fold when the cells were grown in the presence of PCA. Inducers of the pmd operon were found to be PCA and PDC, but PDC was the more effective inducer.Protocatechuate (PCA) is a key intermediate metabolite in the bacterial degradation pathways of various aromatic compounds, including phthalate isomers, vanillate, and hydroxybenzoates. It is known that PCA is degraded via three distinct catabolic pathways, including the PCA 2,3-cleavage (8, 18), PCA 3,4-cleavage (14), and PCA 4,5-cleavage (PCA45) (19,26,27) pathways. Our research group has discovered that 2-pyrone-4,6-dicarboxylic acid (PDC), an intermediate of the PCA45 pathway (Fig. 1A), is useful in the production of biodegradable and high-functional polymers, such as strong adhesives (15,16,30). The production of PDC via the PCA45 pathway from lignin-derived compounds and petrochemical aromatic compounds, including phthalates, would be worthwhile for reducing the environmental load. From this aspect, the catabolic functions of Comamonas sp. strain E6, which is able to utilize phthalate isomers as sole carbon and energy sources via the PCA45 pathway (11, 38), appears to be of importance.The PCA45 pathway was first enzymatically characterized by Kersten et al. (19) and . In this pathway (Fig. 1A), PCA is initially transformed to 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) by PCA 4,5-dioxygenase (4,5-PCD). CHMS is nonenzymatically converted to an intramolecular hemiacetal form and then oxidized by CHMS dehydrogenase. The resulting intermediate, PDC, is hydrolyzed by PDC hydrolase to yield the keto and enol tautomers of 4-oxa...

Novel Tripartite Aromatic Acid Transporter Essential for Terephthalate Uptake in Comamonas sp. Strain E6

“…E6 and KF-1 may employ both the TpiA-TpiB system and an MFS transporter to take up OPA. In regard to PCA uptake, we found an MFS transporter gene, pmdK, in the PCA 4,5-cleavage pathway operon, pmdUKEFD ABC, of E6 (31). The deduced amino acid sequence of E6 PmdK shows 47% identity with the PcaK transporter of 4-hydroxybenzoate/PCA in P. putida (2).…”

“…TPA and its metabolite PCA were detected at 242 nm. In the ESI-MS analysis, mass spectra were obtained using the negative ion mode with the settings specified in a previous study (31). Conversions of TPA by the resting cells of transformants were examined in a 0.5-ml reaction mixture containing 1 mM TPA, cells with an OD 600 of 20, and 50 mM Tris-HCl buffer (pH 7.5).…”

“…A qRT-PCR analysis was carried out using a Fast SYBR green master mix (Applied Biosystems, Foster City, CA) with a StepOne real-time PCR system (Applied Biosystems) according to previously reported methods (31). Primers used for real-time PCR are listed on Table S2 in the supplemental material.…”

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

“…Zinc and Cobalt Dissociation Constant DeterminationApoprotein of either the wild type or variants of GalB was incubated with varying concentrations of either ZnSO 4 or CoCl 2 in a total volume of 600 l of 100 mM HEPES, pH 7.0, and was kept at 15°C for 1 h with constant mixing. Binding reactions were then filtered by ultrafiltration using an Amicon Microcon YM10 to remove enzyme from the solution.…”

Three-Component O -Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6

“…Compounds were detected at 223 nm. In the electrospray ionization-mass spectrometry (ESI-MS) analysis, mass spectra were obtained by using the negative-ion mode with the settings described in a previous study (53).…”