Uncovering the Origins of Selectivity in Non-Heme Iron Dioxygenase-Catalyzed Tropolone Biosynthesis.

Non-heme iron (NHI) enzymes perform diverse oxidative transformations with precise control, which can be challenging to achieve with small molecule catalysts, such as the biosynthesis of tropolone. Among them, Anc3, a reconstructed ancestral α-ketoglutarate (α-KG)-dependent NHI dioxygenase, catalyzes a ring-expansion in fungal tropolone biosynthesis from a cyclohexadienone to afford the tropolone natural product stipitaldehyde (ring-expansion product) alongside 3-hydroxyorcinaldehyde (shunt product). This study reveals how the enzyme environment guides the reaction to the ring-expansion product preferably over the shunt product, where the precise selectivity ratio depends on just a handful of Anc3 residues. In particular, molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) simulations describe how the substrate binds within the NHI active site and can proceed through two distinct mechanisms, ring-expansion or rebound hydroxylation, to yield the two experimentally observed products. Discovery of a linear relationship of Δ