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

• Published in: The FEBS journal Vol. 284; no. 12; pp. 1855 - 1867
• Main Authors: Harada, Ayaka, Kamimura, Naofumi, Takeuchi, Koh, Yu, Hong Yang, Masai, Eiji, Senda, Toshiya
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.06.2017; Federation of European Biochemical Societies
• Subjects: Amino Acid Sequence; Amino acids; Aminomethyltransferase - chemistry; Aromatic compounds; Atomic structure; Biochemistry; Biological evolution; Carbon; Catalysis; Catalytic activity; Cleavage; Crystal structure; Crystallography, X-Ray; Derivatives; Energy consumption; Glycine; GTP-binding protein; Lignin; Metabolism; Models, Molecular; Nucleic acids; Nutrient requirements; Nutrients; Oxidoreductases, O-Demethylating - chemistry; Oxidoreductases, O-Demethylating - metabolism; Oxygen; O‐demethylase; Protein Conformation; Proteins; Sequence Homology, Amino Acid; Similarity; Sphingomonadaceae - enzymology; Substrates; Syk protein; tetrahydrofolate; Tetrahydrofolates - metabolism; Tetrahydrofolic acid; Vanillic Acid - metabolism; crystal structure; lignin; O-demethylase; tetrahydrofolate
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14085

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 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, 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 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 H4folate. 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‐H4folate complex: 5X1IL, LigM‐H4folate‐protocatechuate (PCA) complex (P21212): 5X1M, LigM‐H4folate‐PCA complex (P3121): 5X1N. An O‐demethylase LigM derived from Sphingobium sp. strain SYK‐6 catalyzes O demethylation of vanillate using tetrahydrofolate as a cofactor. While LigM shows an amino acid sequence similarity to the T‐protein, which is involved in the C1‐metabolism in most organisms, the crystal structure of LigM shows that structural characteristics of the active site are distinct from those of T‐protein.