Alteration of the catalytic properties of the epoxyalcohol synthase CYP443D1 (NvEAS) of the starlet sea anemone Nematostella vectensis as a result of a single amino acid substitution

Cytochromes of the P450 superfamily are widespread in nature; they were found in all studied aerobic organisms. Although the degree of similarity between cytochromes P450 of different families is low, all enzymes of this superfamily have similar tertiary structures. In addition, all cytochromes P450, including enzymes of the CYP74 clan, contain substrate recognition sites in their sequences, which form the catalytic center. Initially, CYP74 enzymes were discovered in plants, where they are widespread and play an important role in the lipoxygenase cascade. Later, CYP74-like enzymes of other families were identified in different taxa, including animals. Based on the results of phylogenetic studies, structures, and catalytic mechanisms, they were combined along with the CYP74 family into the CYP74 clan. One of the CYP74 clan enzymes is the epoxyalcohol synthase NvEAS (CYP443D1) of the starlet sea anemone Nematostella vectensis. A mutant form of NvEAS with a P93G substitution, that acquired additional hydroperoxide lyase activity, was obtained by site-directed mutagenesis. Before this work, only the results of site-directed mutagenesis of enzymes of the CYP74 family, but not of the CYP74 clan, were described. Moreover, in this work, the transformation of epoxyalcohol synthase into hydroperoxide lyase is described for the first time. These results confirm the previously stated assumption about the evolution of CYP74 enzymes, namely the epoxyalcohol synthase – hydroperoxide lyase – allene oxide synthase – divinyl ether synthase pathway.