Cyclopropanation and aziridination catalyzed by non-heme iron and 2-oxoglutarate dependent enzymes

Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.