Tomokuni Abe

A Tetrahydrofolate-Dependent O-Demethylase, LigM, Is Crucial for Catabolism of Vanillate and Syringate in Sphingomonas paucimobilis SYK-6

Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.

A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate

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.

Characterization of FerC, a MarR-type transcriptional regulator, involved in transcriptional regulation of the ferulate catabolic operon in Sphingobium sp. strain SYK-6.

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.

Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

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.

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, pmdAIIBII, were isolated from a phthalate-degrading bacterium, Comamonas sp. strain E6. Disruption of pmdB and pmdD in E6, which code for the β 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 pmdBII 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.

Characterization of the Third Glutathione S-Transferase Gene Involved in Enantioselective Cleavage of the β-Aryl Ether by Sphingobium sp. Strain SYK-6

The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the β-aryl ether of (+)-(βS)-α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) and (−)-(βR)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (−)-(βR)-MPHPV, and ligE and ligP alone contributed to the degradation of (−)-(βR)-MPHPV in SYK-6.

Beta-ketoadipic acid and muconolactone production from a lignin-related aromatic compound through the protocatechuate 3,4-metabolic pathway.

In this work, the effects of PcaJ (beta-ketoadipate:succinyl-coenzyme A transferase)- and PcaD (beta-ketoadipate enol-lactone hydrolase)-inactivation on protocatechuic acid metabolism in Pseudomonas putida KT2440 were evaluated. Beta-ketoadipic acid was produced from protocatechuic acid by the inactivation of PcaJ as expected; however, a portion of the produced beta-ketoadipic acid was converted to levulinic acid through a purification step consisting of extraction from the culture and recrystallization. On the other hand, muconolactone was purified from the culture of the PcaD-inactivated mutant of KT2440, although beta-ketoadipate enol-lactone was supposed to be produced because it is the substrate of PcaD. Under aerobic conditions, it has been reported that lignin-related aromatics are metabolized through PCA 2,3- or 3,4- or 4,5-ring cleavage pathways, and muconolactone is an intermediate observed in the metabolism of catechol, not protocatechuic acid. Our results will provide a prospective route to produce muconolactone with a high yield through the protocatechuate-3,4-metabolic pathway.