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 ΔEa 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.