Abstract of the PDB Structure's related Publication:
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 bacteria, resulting in expanded codon-recognition properties. 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.
CmoA is involved in the formation of cmo(5)U. It was annotated as an S-adenosyl-Lmethonin dependent (SAM-dependent) methyltransferase, given its sequence homology with SAM-contianing-enzymes (Byrne et al. 2013 ). Nevertheless, a novel S-adenosyl-S-carboxymethyl-L-homocysteine factor was observed along its sequence in which the donor methyl group is substituted by a carboxymethyl group. This has suggested the reannotation of ComA to SCM-SHA and not SAM (Byrne et al. 2013 ).
CmoA targets tRNAPro. Null mutations of CmoA bring to the accumulation of 5-methoxyuridine (mo5U34) and ho5U34 and the absence of cmo5U34. The presence of these modifications brings to a reduced reading activity of tRNAPro. Indeed, these results suggest that cmoA is directly involved in the carboxymethylation of ho5U34 to cmo5U34 as the final step of a reaction pathway that involves modification of U34 to ho5U34 by unidentified enzymes and hoU34 to mo5U by CmoB (Nasvall et al. 2013 ).