Biochemical Studies of a Cyanobacterial Halogenase Support the Involvement of a Dimetal Cofactor.

Halogenation is a prominent transformation in natural product biosynthesis, with over 5000 halogenated natural products known to date. Biosynthetic pathways accomplish the synthetic challenge of selective halogenation, especially at unactivated sp³ carbon centers, using halogenase enzymes. The halogenase CylC, discovered as part of the cylindrocyclophane (cyl) biosynthetic pathway, performs a highly selective chlorination reaction on an unactivated sp³ carbon center and is proposed to use a dimetal cofactor. Putative dimetal halogenases are widely distributed across cyanobacterial biosynthetic pathways. However, rigorous in vitro biochemical and structural characterization of these enzymes has been challenging. Here, we report additional bioinformatic analyses of putative dimetal halogenases and the biochemical characterization of a newly identified CylC homologue. Site-directed mutagenesis identifies highly conserved putative metal-binding residues, and Mössbauer spectroscopy provides direct evidence for the presence of a diiron cofactor in these halogenases. These insights suggest mechanistic parallels between diiron and mononuclear nonheme iron halogenases, with the potential to guide further characterization and engineering of this unique subfamily of metalloenzymes.