Insertion of 4‑Demethylwyosine in tRNAPhe Catalyzed by the Radical S‑Adenosyl‑l‑methionine Enzyme TYW1 Entails Oxidative Cleavage of Pyruvate to Form CO2

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 tRNAPhe to form 4-demethylwyosine (imG-14) modified tRNAPhe. The fate of C-1 is not known, and either formate or carbon dioxide (CO2) has been proposed. In this study, a coupled assay that transforms either CO2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1-13C1]-pyruvate, 13C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO2 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.