Novel Strategies in Light-driven P450 Enzymes

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Project Summary: Cytochromes P450 are a superfamily of heme-thiolate monooxygenases, found in almost all living organisms, that play an important role in the biosynthesis and biodegradation of endogenous compounds. In humans, P450s are the major enzymes involved in drug metabolism and bioactivation, accounting for 75% of the total metabolism. Harnessing the synthetic potential of these monooxygenases has also led to the recent use of P450 enzymes in late-stage functionalization of various compounds. Our laboratory has developed an efficient light-driven P450 hybrid enzyme system that utilizes the photochemical properties of Ru(II)-diimine photosensitizers covalently attached to strategically positioned non-native single cysteine mutants of their heme domain to perform P450 reactions upon visible light excitation. We recently started to investigate a complementary bimolecular approach to activate P450 enzymes with peroxygenase activity, which is the focus of Aim 1. Using various protein engineering approaches, we are assembling a large and diverse library of light- initiated P450 enzymes with unique photocatalytic activity towards a range of substrates and with human P450-like activities. We have also recently focused on developing chemoenzymatic approaches by marrying the advantages of chemical catalysis with the selectivity of the P450 biocatalysis. We identified a promising substrate anchoring approach to enhance the catalytic activity of P450 enzymes and alter their regioselectivity. In Aim 2, we will capitalize on those strategies to synthesize a library of biphenylalkanoic acid and diphenylamine derivatives, as potential dual fatty acid amide hydrolase (FAAH)/cyclooxygenase (COX) inhibitors to mitigate gastric damages, currently plaguing the use of nonsteroidal anti-inflammatory drugs (NSAIDs). A combination of computer docking simulation and biochemical assays will be used to ascertain their inhibitory activity. In Aim 3, we will apply concepts identified in the substrate engineering approach to develop a new platform for late stage substrate functionalization. Our long-term goal is to employ the libraries of light-driven P450 enzymes combined with the chemoenzymatic and substrate engineering strategies for the development of novel drugs and late stage diversification of lead compounds.