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 that is not easily available to small molecule catalysis, such as the biosynthesis of tropolone. Among them, Anc3, a reconstructed ancestral α-ketoglutarate (α-KG) dependent NHI dioxygenase, catalyzes a ring-expansion in the tropolone biosynthesis from cyclohexadienone to afford tropolone natural product stipitaldehyde (ring-expansion product) alongside 3-hydroxyorcinaldehyde (shunt product). This study reveals how the enzyme environment guides the reaction to ring-expansion product preferably over shunt product, where the precise selectivity ratio depends on just a handful of 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 different Ea values and hydrogen bond distances between Arg191 and the Fe(III)–OH group reveals that inhibition of the rebound hydroxylation step increases selectivity towards 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 ruled out based upon thermodynamic infeasibility. Altogether, the atomistic details and reaction mechanisms delineated in this work have the potential to guide the tuning of reaction pathway in related NHI enzymes for selective oxidation reactions.