PMID:15743951

Citation	Abe, T, Masai, E, Miyauchi, K, Katayama, Y and Fukuda, M (2005) A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6. J. Bacteriol. 187:2030-7
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.
Links	PubMed PMC1064056 Online version:10.1128/JB.187.6.2030-2037.2005
Keywords	Gallic Acid/analogs & derivatives; Gallic Acid/metabolism; Molecular Sequence Data; Mutagenesis; Oxidoreductases, O-Demethylating/genetics; Oxidoreductases, O-Demethylating/metabolism; Sequence Analysis, DNA; Sphingomonas/enzymology; Sphingomonas/genetics; Tetrahydrofolates/metabolism; Vanillic Acid/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PSEPA:Q7WST1	Contributes to	GO:0018487: vanillate O-demethylase (anaerobic) activity	ECO:0000315:		F		Fig. 6A: While the vanillate conversion rate of the cell extract of DKLM incubated with vanillate was strikingly reduced, DDAM (ligM and desA double mutant) cells no longer showed any such activity. These results indicated that only ligM and desA are involved in vanillate O demethylation.	complete CACAO 4991
PSEPA:Q60FX1	Contributes to	GO:0018487: vanillate O-demethylase (anaerobic) activity	ECO:0000315:		F		Fig. 6A: The vanillate conversion rate of the cell extract of DKLM (ligM mutant) incubated with vanillate was strikingly reduced. ligM plays a major role in the O demethylation of vanillate.	complete CACAO 4970

References

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