Reaction pathway engineering converts a radical hydroxylase into a halogenase

Fe^II/α-ketoglutarate (Fe^II/αKG)-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/αKG-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. [Figure not available: see fulltext.].