Discovery of noncanonical cytochrome P450 enzymes in nature

Cytochrome P450s (CYPs) constitute a superfamily of thiolate-ligated heme metalloenzymes principally responsible for the hydroxylation of unactivated C-H bonds. The proximal cysteine is an obligatory and universally conserved residue for the CYP enzyme class. Herein, we challenge this paradigm by systematically identifying noncanonical CYPs (ncCYPs) that do not harbor a proximal cysteine ligand. Our bioinformatic search revealed 20 distinct ncCYP families encoded in diverse microbial genomes with alternative residues at this position. We characterize a native serine-ligated CYP with a high-spin ferric resting state that catalyzes azide reduction and nitrene insertion reactions. Its crystal structure clearly shows a typical CYP fold and a serine alkoxide as a proximal heme ligand. In addition, we report the discovery and characterization of the first native selenocysteine-ligated CYP in nature. Our findings expand the CYP metalloenzyme family and provide opportunities for future enzymatic and biocatalytic discoveries.