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<i>E</i>_a values and hydrogen bond distances between Arg191 and the Fe(III)-OH group reveals that inhibition of the rebound hydroxylation step increases selectivity toward ring-expansion. Our findings suggest that the rebound hydroxylation rate is further tuned through the Fe(III)-OH bond strength, as influenced by specific secondary sphere coordination effects around the active site. These influences are largely orthogonal to the ring-expansion mechanism, which is shown to prefer to proceed through a radical pathway. In addition, a cationic pathway initiated by electron transfer from substrate to iron is shown to be unfavorable based upon thermodynamic considerations. Altogether, the atomistic details and reaction mechanisms delineated in this work have the potential to guide the tuning of the reaction pathway in related NHI enzymes for selective oxidation reactions.