Mechanistic studies of the radical SAM enzyme 4-demethylwyosine synthase reveals the site of hydrogen atom abstraction

TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N -methylguanosine (m 1 G) 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 2 H labeled m 1 G methyl group was used to identify the methyl group of m 1 G as the site of hydrogen atom abstraction by S -adenosyl-L-methionine. [2- 13 C 1 ,3,3,3- 2 H 3 ]-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.