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. The classical P450 reaction is the introduction of an oxygen atom, derived from molecular oxygen, into a substrate unactivated carbon center. The mechanism involves the reductive scission of the O-O bond at the iron center, leading to the formation of a highly oxidative heme radical ferryl species, namely Compound I. Most P450 monooxygenases are characterized by low activity, limited stability, need of an expensive cofactor (NAD(P)H) to reduce the heme iron and general dependence on auxiliary electron carrier proteins called reductases. We propose to develop a novel P450 system that will utilize light and water as the only source of oxygen atom to perform, under inert atmosphere, the selective hydroxylation of various substrate C-H bonds. This system will be used as a human P450 model to facilitate the identification of toxic metabolites early in the drug development process and to enable the diversification of lead compounds through the generation of a broad range of hydroxylated derivatives. The innovative part of this proposal is to replace the reductase by a photosensitizer Ru(II) diimine complex covalently attached to the heme domain. The reactive Compound I will then be generated, under inert atmosphere, via a photo-oxidative route directly from the water bound to the Fe(III) in the resting state of the enzyme rather than through the current reductive mechanism. This approach will enable the control of the electron flow between the photosensitizer and the heme domain while avoiding the rapid deactivation of the protein due to reactive oxygen species. The proposed system will result in enhanced activity of the enzymes towards the selective hydroxylation of substrate C-H bonds.
This proposal aims at developing a novel biocatalyst to selectively insert an oxygen atom into a substrate C-H bond using visible light and water, under inert atmosphere. This system will be used as an innovative model of human P450 enzymes, which are responsible for the bioactivation and biodegradation of 75% of all drugs.
|Spradlin, Jessica; Lee, Diana; Mahadevan, Sruthi et al. (2016) Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies. Biochim Biophys Acta 1864:1732-1738|
|Lam, Quan; Kato, Mallory; Cheruzel, Lionel (2016) Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis. Biochim Biophys Acta 1857:589-97|
|Kato, Mallory; Nguyen, Daniel; Gonzalez, Melissa et al. (2014) Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis. Bioorg Med Chem 22:5687-91|
|Dwaraknath, Sudharsan; Tran, Ngoc-Han; Dao, Thanh et al. (2014) A facile and versatile methodology for cysteine specific labeling of proteins with octahedral polypyridyl dâ¶ metal complexes. J Inorg Biochem 136:154-60|
|Tran, Ngoc-Han; Nguyen, Daniel; Dwaraknath, Sudharsan et al. (2013) An efficient light-driven P450 BM3 biocatalyst. J Am Chem Soc 135:14484-7|
|Tran, Ngoc-Han; Huynh, Ngoc; Chavez, Garrett et al. (2012) A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid. J Inorg Biochem 115:50-6|
|Tran, Ngoc-Han; Huynh, Ngoc; Bui, Thuba et al. (2011) Light-initiated hydroxylation of lauric acid using hybrid P450 BM3 enzymes. Chem Commun (Camb) 47:11936-8|