Mechanism of oxidative ring-closure as part of the hygromycin biosynthesis step by a nonheme iron dioxygenase

Nonheme iron dioxygenases catalyze vital reactions for biosystems including the biosynthesis of antibiotics. One such enzyme, namely hygromycin (HygX), performs an oxidative ring-closure reaction to form an ortho-ester product, which is a relevant reaction step in drug synthesis and biotechnology. To understand the selective reaction mechanism of oxidative ring-closure to form ortho-ester products, we investigated the catalytic reaction mechanism of HygX leading to various products. Large active site cluster models were set-up and various pathways for substrate activation have been calculated. The work identifies a high-valent iron(IV)-oxo species in the quintet spin state as the active oxidant that selectively abstracts a proton of an alcohol group of the substrate, which is followed by a hydrogen atom abstraction from a tertiary CH group and rapid electron transfer. The latter formed biradical intermediate rearranges to form the desaturated ring-closed product. The calculations show that an active site Lys residue donates positive charge to the metal-oxo group and guides the reaction to a chemoselective desaturation pathway.