Proposed reaction mechanism for the biosynthetic pathway of mnm5U34. In the first step, the MnmE/MnmG protein complex catalyzes the fusion of formyl group from 5-formyl tetrahydrofolate (THF) and the wobble uridine (U34) of tRNA. The amino group of glycine attacks the carbonyl group of formyl-tRNA to yield a Schiff base, which is reduced by MnmG-bound FADH. Carboxymethylaminomethyl uridine (cmnm5U) is oxidized between Cα-N bond by MnmC1-bound FAD. The resulting imine intermediate is non-enzymatically hydrolyzed to yield aminomethyl uridine (nm5U), which is then methylated by SAM in the MnmC2 active site.