Exploration of CYP4B1 Substrate Promiscuity Across Three Species

Enzymes of the cytochrome P450 monooxygenase family 4 (CYP4) in mammals are generally involved either in endobiotic metabolism (e.g., acting on fatty acids or eicosanoids), or the modification of xenobiotics including therapeutic drugs. CYP4B1 is special, as it is an enigmatic enzyme acting at the interface between xenobiotic and endobiotic metabolism. However, a systematic analysis of CYP4B1’s substrate scope has not yet been reported. Herein, a three-step approach to identify novel substrates for three CYP4B1 orthologs (namely from rabbits, green monkeys, and mouse lemurs) is described. First, screening of a library containing 502 natural products revealed potential novel substrate groups for CYP4B1. Second, based on these results, a systematic library was defined consisting of 63 compounds representing 10 compound groups. Third, in vitro conversion of these compounds by CYP4B1 and identification of conversion products were conducted, supported by in silico docking, allowing the prediction of binding probabilities and potential oxidation sites. We report five new substrate groups (acyclic, monocyclic and bicyclic terpenoids, stilbenoids, and vanilloids), twenty-eight new substrates (inter alia capsaicin, gingerol, genistein, stilbene, myristicin, thioanisole), and two new reaction types for CYP4B1 (S-oxidation, O-demethylation). Consequently, CYP4B1 is a far more promiscuous enzyme than previously thought.