Mechanistic Studies of the Radical S-Adenosyl‑l‑methionine Enzyme 4‑Demethylwyosine Synthase Reveal the Site of Hydrogen Atom Abstraction

TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m1G) with carbons 2 and 3 of pyruvate. In this study, labeled transfer ribonucleic acid (tRNA) and pyruvate were utilized to determine the site of hydrogen atom abstraction and regiochemistry of the pyruvate addition. tRNA containing a 2H-labeled m1G methyl group was used to identify the methyl group of m1G as the site of hydrogen atom abstraction by 5′-deoxyadenosyl radical. [2-13C1-3,3,3-2H3]­Pyruvate was used to demonstrate retention of all the pyruvate protons, indicating that C2 of pyruvate forms the bridging carbon of the imidazoline ring and C3 the methyl.