Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function

Abstract

Members of the SAM-dependent methyltransferase superfamily are involved in the modification of wobble uridine to 5-oxacetyl uridine in Gram-negative bacteria; CmoA converts SAM to carboxy-SAM (Cx-SAM; a metabolite that was unknown previously), and CmoB uses Cx-SAM to convert 5-hydroxyuridine to 5-oxyacetyl uridine in tRNA. Many of the small molecules that are the ultimate mediators of the cell's processes remain undescribed and unreported, as their identities may not emerge from conventional techniques that chart metabolic pathways. Jungwook Kim et al. have identified one such molecule, carboxy-S-adenosyl-L-methionine (Cx-SAM). The previously unknown metabolite was found during a structural study of the Escherichia coli enzyme CmoA, a member of the SAM-dependent methyltransferase superfamily. Cx-SAM was buried in the active site of CmoA. The authors have determined the biosynthetic pathway of Cx-SAM, which proceeds through an unusual reactive ylide intermediate, and show that the metabolite has a role in tRNA modification in a wide range of bacteria. This work highlights the potential of structural genomics for the discovery of novel metabolites and pathways. The identification of novel metabolites and the characterization of their biological functions are major challenges in biology. X-ray crystallography can reveal unanticipated ligands that persist through purification and crystallization. These adventitious protein–ligand complexes provide insights into new activities, pathways and regulatory mechanisms. We describe a new metabolite, carboxy-S-adenosyl-l-methionine (Cx-SAM), its biosynthetic pathway and its role in transfer RNA modification. The structure of CmoA, a member of the SAM-dependent methyltransferase superfamily, revealed a ligand consistent with Cx-SAM in the catalytic site. Mechanistic analyses showed an unprecedented role for prephenate as the carboxyl donor and the involvement of a unique ylide intermediate as the carboxyl acceptor in the CmoA-mediated conversion of SAM to Cx-SAM. A second member of the SAM-dependent methyltransferase superfamily, CmoB, recognizes Cx-SAM and acts as a carboxymethyltransferase to convert 5-hydroxyuridine into 5-oxyacetyl uridine at the wobble position of multiple tRNAs in Gram-negative bacteria1, resulting in expanded codon-recognition properties2,3. CmoA and CmoB represent the first documented synthase and transferase for Cx-SAM. These findings reveal new functional diversity in the SAM-dependent methyltransferase superfamily and expand the metabolic and biological contributions of SAM-based biochemistry. These discoveries highlight the value of structural genomics approaches in identifying ligands within the context of their physiologically relevant macromolecular binding partners, and in revealing their functions.