Publications

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

  Abe T., Masai E., Miyauchi K., Katayama Y., Fukuda M.

  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 ... >> More

  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. << Less

  J. Bacteriol. 187:2030-2037(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The crystal structure of a new O-demethylase from Sphingobium sp. strain SYK-6.

  Harada A., Kamimura N., Takeuchi K., Yu H.Y., Masai E., Senda T.

  In the cell, tetrahydrofolate (H₄ folate) derivatives with a C1 unit are utilized in various ways, such as for the synthesis of amino acids and nucleic acids. While H₄ folate derivatives with the C1 unit are typically produced in the glycine cleavage system, Sphingobium sp. s ... >> More

  In the cell, tetrahydrofolate (H₄ folate) derivatives with a C1 unit are utilized in various ways, such as for the synthesis of amino acids and nucleic acids. While H₄ folate derivatives with the C1 unit are typically produced in the glycine cleavage system, Sphingobium sp. strain SYK-6, which can utilize lignin-derived aromatic compounds as a sole source of carbon and energy, lacks this pathway, probably due to its unique nutrient requirements. In this bacterium, H₄ folate-dependent O-demethylases in catabolic pathways for lignin-derived aromatic compounds seem to be involved in the C1 metabolism. LigM is one of the O-demethylases and catalyzes a C1-unit transfer from vanillate (VNL) to H₄ folate. As the primary structure of LigM shows a similarity to T-protein in the glycine cleavage system, we hypothesized that LigM has evolved from T-protein, acquiring its unique biochemical and biological functions. To prove this hypothesis, structure-based understanding of its catalytic reaction is essential. Here, we determined the crystal structure of LigM in apo form and in complex with substrates and H₄ folate. These crystal structures showed that the overall structure of LigM is similar to T-protein, but LigM has a few distinct characteristics, particularly in the active site. Structure-based mutational analysis revealed that His60 and Tyr247, which are not conserved in T-protein, are essential to the catalytic activity of LigM and their interactions with the oxygen atom in the methoxy group of VNL seem to facilitate a methyl moiety (C1-unit) transfer to H₄ folate. Taken together, our structural data suggest that LigM has evolved divergently from T-protein.<h4>Databases</h4>All atomic coordinates of the crystal structures determined in this study have been deposited to PDB. LigM: 5X1I, LigM-VNL complex: 5X1J, LigM-3-O-methylgallate complex: 5X1K, LigM-H₄ folate complex: 5X1IL, LigM-H₄ folate-protocatechuate (PCA) complex (P2₁ 2₁ 2): 5X1M, LigM-H₄ folate-PCA complex (P3₁ 21): 5X1N. << Less

  FEBS J. 284:1855-1867(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of aryl O-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism.

  Kohler A.C., Mills M.J.L., Adams P.D., Simmons B.A., Sale K.L.

  Some strains of soil and marine bacteria have evolved intricate metabolic pathways for using environmentally derived aromatics as a carbon source. Many of these metabolic pathways go through intermediates such as vanillate, 3-<i>O</i>-methylgallate, and syringate. Demethylation of these compounds ... >> More

  Some strains of soil and marine bacteria have evolved intricate metabolic pathways for using environmentally derived aromatics as a carbon source. Many of these metabolic pathways go through intermediates such as vanillate, 3-<i>O</i>-methylgallate, and syringate. Demethylation of these compounds is essential for downstream aryl modification, ring opening, and subsequent assimilation of these compounds into the tricarboxylic acid (TCA) cycle, and, correspondingly, there are a variety of associated aryl demethylase systems that vary in complexity. Intriguingly, only a basic understanding of the least complex system, the tetrahydrofolate-dependent aryl demethylase LigM from <i>Sphingomonas paucimobilis</i>, a bacterial strain that metabolizes lignin-derived aromatics, was previously available. LigM-catalyzed demethylation enables further modification and ring opening of the single-ring aromatics vanillate and 3-<i>O</i>-methylgallate, which are common byproducts of biofuel production. Here, we characterize aryl <i>O</i>-demethylation by LigM and report its 1.81-Å crystal structure, revealing a unique demethylase fold and a canonical folate-binding domain. Structural homology and geometry optimization calculations enabled the identification of LigM's tetrahydrofolate-binding site and protein-folate interactions. Computationally guided mutagenesis and kinetic analyses allowed the identification of the enzyme's aryl-binding site location and determination of its unique, catalytic tyrosine-dependent reaction mechanism. This work defines LigM as a distinct demethylase, both structurally and functionally, and provides insight into demethylation and its reaction requirements. These results afford the mechanistic details required for efficient utilization of LigM as a tool for aryl <i>O</i>-demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds. << Less

  Proc. Natl. Acad. Sci. U.S.A. 114:E3205-E3214(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.