Engineering the Reaction Pathway of a Non-heme Iron Oxygenase Using Ancestral Sequence Reconstruction

Non-heme iron (FeII), α-ketoglutarate
(α-KG)-dependent
oxygenases are a family of enzymes that catalyze an array of transformations
that cascade forward after the formation of radical intermediates.
Achieving control over the reaction pathway is highly valuable and
a necessary step toward broadening the applications of these biocatalysts.
Numerous approaches have been used to engineer the reaction pathway
of FeII/α-KG-dependent enzymes, including site-directed
mutagenesis, DNA shuffling, and site-saturation mutagenesis, among
others. Herein, we showcase a novel ancestral sequence reconstruction
(ASR)-guided strategy in which evolutionary information is used to
pinpoint the residues critical for controlling different reaction
pathways. Following this, a combinatorial site-directed mutagenesis
approach was used to quickly evaluate the importance of each residue.
These results were validated using a DNA shuffling strategy and through
quantum mechanical/molecular mechanical (QM/MM) simulations. Using
this approach, we identified a set of active site residues together
with a key hydrogen bond between the substrate and an active site
residue, which are crucial for dictating the dominant reaction pathway.
Ultimately, we successfully converted both extant and ancestral enzymes
that perform benzylic hydroxylation into variants that can catalyze
an oxidative ring-expansion reaction, showcasing the potential of
utilizing ASR to accelerate the reaction pathway engineering within
enzyme families that share common structural and mechanistic features.