Insertion of 4-Demethylwyosine in tRNA Phe Catalyzed by the Radical S -Adenosyl-l-methionine Enzyme TYW1 Entails Oxidative Cleavage of Pyruvate to Form CO 2 .

The radical S -adenosyl-l-methionine (SAM) enzyme TYW1 catalyzes the condensation of C-2 and C-3 atoms of pyruvate with N -methylguanosine containing tRNA Phe to form 4-demethylwyosine (imG-14) modified tRNA Phe . The fate of C-1 is not known, and either formate or carbon dioxide (CO 2 ) has been proposed. In this study, a coupled assay that transforms either CO 2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1- 13 C 1 ]-pyruvate, 13 C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO 2 and not formate to OAA. These findings are discussed in the context of the cofactor content of TYW1 and a new role for the auxiliary cluster in catalyzing the oxidative cleavage of C-1-C-2 bond of pyruvate in the catalytic cycle of TYW1.