Rapid probing of the reactivity of P450 monooxygenases from the CYP116B subfamily using a substrate-based method

Developing a detailed understanding of the reactivity of self-sufficient Type IV P450 monooxygenases, four types of O-methylated substrates were designed as probes, including monoterpenes, cycloalkanes, aromatic compounds and steroids, and the efficiency of their oxyfunction was determined using a colorimetric assay which was based on the reaction between the enzymatic demethylation product, formaldehyde, and Purpald dye. The activity-based fingerprints of new P450RpMO, P450ArMO and P450CtMO (CYP116B members) indicated that CYP116B P450s preferentially oxidize substrates with aromatic components. Moreover, the hydroxylated products were detected based on the preference results. This rapid and efficient strategy, when coupled with GCMS, enables the exploration of the reactivity of other CYP116B members.