Reaction pathway engineering converts a radical hydroxylase into a halogenase

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Synthetic Chemistry

Background:

• Iron(II)/α-ketoglutarate (FeII/αKG)-dependent enzymes are valuable for C-H bond functionalization.
• Limited availability of radical halogenases restricts their synthetic applications.

Purpose of the Study:

• To engineer FeII/αKG-dependent hydroxylases into active halogenases.
• To expand the scope of enzymatic halogenation reactions.

Main Methods:

• DNA shuffling of a halogenase-hydroxylase pair.
• High-throughput in vivo fluorescent screening coupled with an alkyne-producing pathway.
• Protein engineering guided by structural insights and variant sequencing.

Main Results:

• Discovery of novel active halogenases from engineered libraries.
• Successful conversion of a hydroxylase into a halogenase with high activity and selectivity.
• Demonstration of a strategy to repurpose hydroxylases for halogenation.

Conclusions:

• Enzyme engineering can successfully transform hydroxylases into effective halogenases.
• This approach broadens the toolkit for biocatalytic halogenation.
• Harnessing hydroxylases offers a promising avenue for novel halogenation chemistry.