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

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.

Databases: 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.