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

Abstract

Identifying novel metabolites and characterizing their biological functions are major challenges of the post-genomic era. X-ray crystallography can reveal unanticipated ligands which 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-adenosylmethionine (Cx-SAM), its biosynthetic pathway and its role in tRNA modification. The structure of CmoA, a member of the SAM-dependent methyltransferase superfamily, revealed a ligand in the catalytic site consistent with Cx-SAM. Mechanistic analyses demonstrated 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 (ho5U) into 5-oxyacetyl uridine (cmo5U) 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 for identifying ligands in the context of their physiologically relevant macromolecular binding partners and for aiding in functional assignment.

Structure of dihydrouridine synthase C (DusC) fromEscherichia coli

“…Further studies are currently under way in our laboratory to identify the adapter molecule in order to gain further insight into the mechanism underlying D modification. Carboxy-S-adenosyl-l-methionine has recently been identified as a novel cofactor molecule for tRNAmodification enzymes based on high-resolution crystal structures (Byrne et al, 2013;Kim et al, 2013). Determining the high-resolution structure of Dus enzymes may be helpful in identifying the adapter molecule.…”

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative

“…Experience clearly demonstrates the challenges in securing resources to work in unexplored areas owing to a lack of compelling hypotheses. Without discovery-driven efforts, large amounts of biology will continue to remain obscure, and it will not be possible to systemically examine the structural features of full proteomes, which undoubtedly would lead to unexpected functional and biological insights (7)(8)(9).…”

Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism

“…It is also instructive to compare the chemistry carried out by Elp3 with the chemistry carried out by CmoA and CmoB, which also perform tRNA wobble uridine modification at C5 in Gram-negative bacteria 16 . Both systems transfer a carboxymethyl group from a cofactor to U34, but they employ different chemistry.…”