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Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H 4 folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H 4 folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H 4 folate, forming 5-methyl-H 4 folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H 4 folate. The possible role of ligH is discussed.Lignin is the most abundant aromatic compound in nature, and the utilization of lignin for production of chemicals has been expected. One of the practical procedures for utilizing lignin is its conversion to valuable intermediate metabolites using the microbial lignin degradation enzyme systems (22). It is known that the degradation of native lignin is initiated by the attack by lignin peroxidase, manganese peroxidase, and laccase secreted by white rot fungi (14), and bacteria contribute to the process of mineralization of the abundant lignin-derived compounds found in soil (44, 47). In microbial degradation of lignin-derived compounds, vanillate and syringate are the important intermediate metabolites. Sphingomonas paucimobilis SYK-6 is able to utilize these compounds and various ligninderived biaryls as the sole source of carbon and energy (20-22, 29, 30). Vanillate and syringate are O demethylated by this strain to produce protocatechuate (PCA) and 3-O-metylgallate (3MGA), respectively. PCA is further degraded through the PCA 4,5-cleavage pathway. In contrast, it has been found that 3MGA is degraded via multiple pat...

Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon.Protocatechuate (PCA) is one of the key intermediate metabolites in the microbial catabolic pathways for various aromatic compounds, including phthalates, hydroxybenzoates, and lignin-derived aromatic compounds such as vanillate and ferulate. It is known that the aromatic ring fission of PCA is catalyzed by one of the three distinct dioxygenases PCA 3,4-dioxygenase (26), PCA 4,5-dioxygenase (36, 41), and PCA 2,3-dioxygenase (7). In the PCA 3,4-cleavage pathway, PCA is converted into 2-carboxy-cis,cis-muconate by the reaction catalyzed by PCA 3,4-dioxygenase, and the catabolic pathway for its product (␤-ketoadipate pathway) has been reported in many bacteria (24,26). In the case of the PCA 4,5-cleavage pathway, PCA is cleaved by PCA 4,5-dioxygenase to yield 4-carboxy-2-hydroxymuconate-6-semialdehyde, and then the product is degraded to 2-pyrone-4,6-dicarboxylate, 4-oxalomesaconate, and 4-carboxy-4-hydroxy-2-oxoadipate before entering the Krebs cycle (36). The genes and enzymes involved in this pathway have been recently characterized for several bacteria, such as Sphingobium (Sphingomonas) (36), Comamonas (47), Pseudomonas (35), and Arthrobacter (13) strains. On the other hand, no genetic information on the PCA 2,3-cleavage pathway has been reported since the finding of this pathway in some bacilli (7,8).In 1979, Crawford et al. reported the PCA 2,3-cleavage pathway of a 4-hydroxybenzoate (4HB) degrader, Paenibacillus sp. ...

Sphingobium sp. strain SYK-6 is able to degrade various lignin-derived aromatic compounds including ferulate, vanillate, and syringate. In the SYK-6 cells, ferulate is converted to vanillin and acetyl-coenzyme A (acetyl-CoA) through the reactions catalyzed by feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase encoded by ferA and ferB, respectively. Here, we characterized the transcriptional regulation of ferBA controlled by a MarR-type transcriptional regulator, FerC. The ferC gene is located upstream of ferB. Reverse transcription (RT)-PCR analysis suggested that the ferBA genes form an operon. Quantitative RT-PCR analyses of SYK-6 and its mutant cells revealed that the transcription of the ferBA operon is negatively regulated by FerC, and feruloyl-CoA was identified as an inducer. The transcription start site of ferB was mapped at 30 nucleotides upstream from the ferB initiation codon. Purified His-tagged FerC bound to the ferC-ferB intergenic region. This region contains an inverted repeat sequence, which overlaps with a part of the -10 sequence and the transcriptional start site of ferB. The binding of FerC to the operator sequence was inhibited by the addition of feruloyl-CoA, indicating that FerC interacts with feruloyl-CoA as an effector molecule. Furthermore, hydroxycinnamoyl-CoAs, including p-coumaroyl-CoA, caffeoyl-CoA, and sinapoyl-CoA also acted as effector.

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...