TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m<sup>1</sup>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 <sup>2</sup>H-labeled m<sup>1</sup>G methyl group was used to identify the methyl group of m<sup>1</sup>G as the site of hydrogen atom abstraction by 5′-deoxyadenosyl radical. [2-<sup>13</sup>C<inf>1</inf>-3,3,3-<sup>2</sup>H<inf>3</inf>]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.
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