Reaction pathway engineering converts a radical hydroxylase into a halogenase

Fe-II/alpha-ketoglutarate (Fe-II/alpha KG)-dependent enzymes offer a promising biocatalytic platform for halogenation chemistry owing to their ability to functionalize unactivated C-H bonds. However, relatively few radical halogenases have been identified to date, limiting their synthetic utility. Here, we report a strategy to expand the palette of enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity. This approach could allow us to tap the broader class of Fe-II/alpha KG-dependent hydroxylases as catalysts by their conversion to halogenases. Toward this goal, we discovered active halogenases from a DNA shuffle library generated from a halogenase-hydroxylase pair using a high-throughput in vivo fluorescent screen coupled to an alkyne-producing biosynthetic pathway. Insights from sequencing halogenation-active variants along with the crystal structure of the hydroxylase enabled engineering of a hydroxylase to perform halogenation with comparable activity and higher selectivity than the wild-type halogenase, showcasing the potential of harnessing hydroxylases for biocatalytic halogenation.

Automated summary

Researchers have developed a strategy to expand enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity. They discovered active halogenases from a DNA shuffle library and engineered a hydroxylase to perform halogenation with comparable activity and higher selectivity than the wild-type halogenase, showcasing the potential of harnessing hydroxylases for biocatalytic halogenation.