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

Area of Science:

• Biocatalysis and Enzyme Engineering
• Protein Evolution and Design
• Chemical Biology

Background:

• Non-heme iron (FeII) and α-ketoglutarate (α-KG)-dependent oxygenases are versatile enzymes catalyzing radical-mediated transformations.
• Controlling the reaction pathways of these enzymes is crucial for expanding their biocatalytic applications.
• Traditional engineering methods like site-directed mutagenesis and DNA shuffling have limitations in pinpointing critical residues.

Purpose of the Study:

• To develop and showcase a novel ancestral sequence reconstruction (ASR)-guided strategy for engineering FeII/α-KG-dependent oxygenase reaction pathways.
• To identify critical residues and interactions that dictate enzyme reaction pathways using evolutionary information.
• To convert enzymes performing benzylic hydroxylation into variants capable of oxidative ring-expansion reactions.

Main Methods:

• Ancestral sequence reconstruction (ASR) to identify evolutionarily conserved residues important for pathway control.
• Combinatorial site-directed mutagenesis to rapidly assess the impact of identified residues.
• Validation through DNA shuffling and quantum mechanical/molecular mechanical (QM/MM) simulations.

Main Results:

• Identified specific active site residues and a key substrate-active site hydrogen bond crucial for determining the dominant reaction pathway.
• Successfully engineered both extant and ancestral enzymes to switch from benzylic hydroxylation to oxidative ring-expansion.
• Demonstrated the efficacy of ASR in accelerating enzyme pathway engineering by leveraging evolutionary insights.

Conclusions:

• ASR is a powerful tool for pinpointing critical residues that control enzyme reaction pathways.
• The identified residues and hydrogen bond are key determinants for directing the catalytic outcome of FeII/α-KG-dependent oxygenases.
• This ASR-guided approach significantly accelerates the engineering of biocatalysts for novel chemical transformations.