Exquisite Complex Reaction Cascade in the Natural 1,2,4-Triazine Assembly

1,2,4-Triazine ring is a scaffold widely found in biologically active compounds, but how nature makes it remains enigmatic. In this study, we unveil the complex enzymatic and nonenzymatic cascade reactions that assemble the 1,2,4-triazine moiety found in the structures of the natural products pseudoiodinine and toxoflavin. Through biochemical studies, isotope labeling, and the application of substrate analogues, we propose a plausible pathway for the 1,2,4-triazine assembly from a common precursor in riboflavin biosynthesis. This process involves four two-electron oxidation steps, C–N bond formation, decarboxylation, and the N–N bond forming step catalyzed by a metal-dependent WD40-repeat (WDR) protein. This study thus not only provides the first biocatalytic route for the 1,2,4-triazine assembly but also identifies a previously unrecognized catalytic role of a large WDR protein family.