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

In the cell, tetrahydrofolate (H4folate) derivatives with a C1 unit are utilized in various ways, such as for the synthesis of amino acids and nucleic acids. While H4folate derivatives with the C1 unit are typically produced in the glycine cleavage system, Sphingobium sp. strain SYK-6, which can utilize ligninderived aromatic compounds as a sole source of carbon and energy, lacks this pathway, probably due to its unique nutrient requirements. In this bacterium, H4folate-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 H4folate. 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 H4folate. 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 Tprotein, 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 H4folate. Taken together, our structural data suggest that LigM has evolved divergently from T-protein.