Dataset Information

A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6.

ABSTRACT: 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.

SUBMITTER: Abe T

PROVIDER: S-EPMC1064056 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6.

Abe Tomokuni T Masai Eiji E Miyauchi Keisuke K Katayama Yoshihiro Y Fukuda Masao M

Journal of bacteriology 20050301 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 c ...[more]

PMID: 15743951

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Project description:Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX.IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.

Project description:Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells. A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders. On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of E. coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively. p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5'-dehydrodivanillic acid. The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase/lyase activity with a substrate specificity similar to that of FerB. Insertional inactivation of each fer gene in S. paucimobilis SYK-6 suggested that the ferA gene is essential and that ferB and ferB2 genes are involved in ferulic acid degradation.

Dataset Information

A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6.

Publications

A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6.

Similar Datasets

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