Nature, published online 15 May 2013; doi:10.1038/nature12180

Credit: JUNGWOOK KIM

Post-transcriptional tRNA modifications have roles in tRNA stability and in ribosomal decoding. For instance, Gram-negative bacteria frequently contain 5-oxyacetyl uridine (cmo5U) bases in the anticodon wobble position to read four degenerate codons. cmo5U is derived from 5-hydroxyuridine (ho5U) in a process that is dependent on chorismate biosynthesis and CmoA and CmoB, two enzymes in the S-adenosyl-L-methionine (SAM)-dependent methyltransferase superfamily. Kim et al. now identify a new SAM metabolite, carboxy-S-adenosyl-L-methionine (Cx-SAM) and show that it is made by CmoA and used by CmoB for the biosynthesis of cmo5U. An X-ray crystal structure of CmoA from Escherichia coli revealed electron density consistent with a modified SAM moiety. Isolation and LC/MS analysis identified the metabolite as Cx-SAM. Biochemical characterization showed that CmoA catalyzes the conversion of SAM to Cx-SAM and is dependent on prephenate, a chorismate metabolite. 13C labeling showed that the carboxyl group of Cx-SAM is derived from carbon dioxide generated at the CmoA active site by decarboxylation of prephenate. Deuterium labeling of SAM revealed that prephenate also performs a substrate-assisted catalytic role by generating a general base required for formation of the stabilized carbanion that ultimately receives the carboxyl functionality. Finally, the authors demonstrated that CmoB uses Cx-SAM to catalyze the in vitro carboxymethylation of ho5U-modified RNA. In addition to elucidating the biosynthesis of cmo5U, the study outlines a structure-guided approach to metabolite discovery.