Structural and Functional Analysis of SAM-Dependent N-Methyltransferases Involved in Ovoselenol and Ovothiol Biosynthesis

Thio/selenoimidazole N-pi-methyltransferases are an emerging family of enzymes responsible for catalyzing the final enzymatic step in the biosynthesis of ovothiol and ovoselenol, S/Se-containing histidine-derived antioxidants. These unique enzymes are widespread among prokaryotes yet bear only marginal sequence similarity to other known methyltransferases. Likewise, little is known about the structural determinants of their reactivities. Here we report the first ligand-bound X-ray crystal structures of this family, including OvsM from the ovoselenol pathway as well as a member of a previously unknown clade of standalone ovothiol-biosynthetic N-pi-methyltransferases, which we have designated OvoM. Unlike previously reported ovothiol methyltransferases, which are fused as a C-terminal domain to the sulfoxide synthase OvoA, OvoMs are discrete enzymes and function independently. Comparative structural analyses of OvsM and OvoM reveal several conserved, ligand-induced tertiary and secondary structure changes, and suggest that similar conformational changes may apply to dual-domain OvoA enzymes. Mutagenesis experiments support a model in which the rearrangement of OvoA’s two domains facilitates substrate recognition through interaction with a key Tyr residue located within the domain linker. Furthermore, biochemical experiments highlight the essential role of an active site Asp residue, which likely functions as a catalytic base in the SN2-like nucleophilic substitution reaction catalyzed by these enzymes.