Molecular Basis of α-Glycine C-H Activation by a Nonheme Fe(II)/2-Oxoglutarate Dioxygenase

Nonheme Fe(II)/2-oxoglutarate (Fe/2-OG) dioxygenases carry out a broad range of oxidative reactions, yet α-hydroxylation of glycyl residues remains exceedingly rare. Here, we demonstrate that the Fe/2-OG enzyme MysH from Nostoc linckia performs this unusual chemistry during mycosporine-like amino acid biosynthesis, converting mono- and disubstituted precursors into palythines and revealing unexpected substrate tolerance. Kinetic isotope effects, detection of a transient hydroxylated intermediate, and glyoxylate byproduct formation support an α-hydroxylation-initiated mechanism. High-resolution crystal structures, complemented by molecular docking, molecular dynamics simulations, and site-directed mutagenesis, define an active-site architecture that positions the glycyl substrate in a near-transition-state geometry. Hybrid QM/MM calculations reveal a low-barrier hydrogen-atom-transfer step followed by hydroxyl rebound and implicate a conserved Trp125 in an electron-transfer network that lowers the activation barrier. Together, these findings establish a mechanistic framework for protein-directed α-glycine C-H activation by nonheme iron enzymes and provide a blueprint for engineering Fe/2-OG dioxygenases to expand the chemical diversity of mycosporines and related natural products.