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.

FIG. 1.

Proposed O demethylation system linked with H₄folate-mediated C₁ metabolism in S. paucimobilis SYK-6. The reactions indicated by dashed arrows have not been confirmed. Abbreviations: PCA, protocatechuate; 3MGA, 3-O-methylgallate; PDC, 2-pyrone-4,6-dicarboxylate; OMA, 4-oxalomesaconate; CHMOD, 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate; H₄folate, tetrahydrofolate.

FIG. 2.

SDS-PAGE analysis of LigM produced in E. coli BL21(DE3). Proteins (20 μg) were separated in an SDS-12% polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: 1, molecular size markers; 2, crude extract of E. coli harboring pET21a(+); 3, crude extract of E. coli harboring pG-KJE7; 4, crude extract of E. coli harboring pELM; 5, SDS-solubilized cells of E. coli harboring pELM; 6, crude extract of E. coli harboring pELM and pG-KJE7. Molecular masses are given on the left.

FIG. 3.

Identification of reaction products from vanillate and 3MGA catalyzed by LigM. The cell extract of E. coli BL21(DE3) harboring pELM and pG-KJE7 (1 mg of protein/ml) was incubated with 500 μM vanillate or 3MGA in the presence of 1 mM H₄folate. (A and B) Gas chromatograms of the TMS derivative of the reaction product from vanillate at start and after 10 min of incubation, respectively. (D and E) Gas chromatograms of the TMS derivative of the reaction product from 3MGA at start and after 10 min of incubation, respectively. (C and F) Mass spectra of the TMS derivatives of compounds I and II, respectively.

FIG. 4.

Identification of C₁-H₄folate generated by O demethylation of vanillate catalyzed by LigM. The cell extract of E. coli BL21(DE3) harboring pELM and pG-KJE7 (1 mg of protein/ml) was incubated with 5 mM vanillate and H₄folate. The results shown are negative-ion ESI-MS spectra of the reaction mixtures after 30 min of incubation without (A) or with (B) the enzyme.

FIG. 5.

Disruption of ligM and desA in SYK-6. (A) Southern hybridization analysis of insertion mutants. Lanes: 1, 3, 5, and 7, total DNAs of SYK-6 digested with SmaI; 2 and 4, total DNAs of DKLM digested with SmaI; 6 and 8, total DNAs of DDAM digested with SmaI. The 2.8-kb Eco47III fragment carrying ligM (lanes 1, 2, 5, and 6), the 1.3-kb EcoRV fragment carrying kan (lanes 3 and 4), and the 1.0-kb BspHI fragment carrying bla (lanes 7 and 8) were used as probes. (B and C) Growth on vanillate (B) and syringate (C) of SYK-6 (circles), DKLM (squares), DKDA (triangles), and DDAM (cross). These strains were grown in 10 ml of W medium containing 10 mM vanillate or syringate. Each value is the average ± standard deviation (error bars) of three independent experiments.

FIG. 6.

O-demethylase activities of strains DKLM, DKDA, and DDAM. The time courses of degradation of vanillate (A), syringate (B), and 3MGA (C) by cell extracts (2 mg of protein/ml) of SYK-6 (circles), DKLM (squares), DKDA (triangles), and DDAM (cross) incubated with 10 mM vanillate (A) or syringate (B and C) are shown. Each cell extract was incubated with 250 μM vanillate, syringate, or 3MGA in the presence of 1 mM H₄folate under anaerobic conditions. HPLC was used to monitor the time course of substrate removal.