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 selective introduction of an oxygen atom, derived from molecular dioxygen, into a substrate unactivated carbon center. The mechanism involves the consumption of two reducing equivalents and the activation of dioxygen at the heme center, leading to the formation of a highly oxidative porphyrin radical ferryl species, namely Compound I. Most P450 monooxygenases are characterized by low activity, limited stability, need of an expensive cofactor (NAD(P)H) and general dependence on auxiliary electron carrier proteins called reductases. We have developed an efficient light-driven P450 BM3 hybrid enzyme system that circumvents the use of the reductase and the expensive NADPH cofactor. This approach utilizes the photochemical properties of Ru(II) photosensitizers covalently attached to strategically positioned non-native single cysteine residues of P450 BM3 heme domain mutants able to perform P450 reactions upon light activation. The high photocatalytic activity and initial reaction rates obtained with the optimized hybrid P450 BM3 enzymes validated our light- activated approach and lays the foundation for the current proposal. We are planning on solving the crystal structure of the highly active hybrid enzyme (Aim 1) in order to understand the factors controlling the efficient electron transfer as well as to improve the design of future hybrd enzymes. We will also take advantage of the unique properties of the hybrid enzymes to study P450 protein interactions thought to play an important role in human enzyme activity (Aim 2). The last two aims will be dedicated to expanding the scope of the light- activated approach using P450 BM3 mutants able to generate various human drug metabolites (Aim 3) and using a mammalian P450 enzyme, CYP2B4, known to hydroxylate bulky hydrophobic xenobiotics (Aim 4). This light-activated P450 enzyme approach 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.

Public Health Relevance

This proposal aims at developing light-driven hybrid P450 enzymes to produce valuable human drug metabolites upon visible light excitation. 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Continuance Award (SC3)
Project #
5SC3GM095415-07
Application #
9249069
Study Section
Special Emphasis Panel (ZGM1-TWD-3 (SC))
Program Officer
Alexander, Rashada
Project Start
2011-03-01
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
7
Fiscal Year
2017
Total Cost
$97,537
Indirect Cost
$30,037
Name
San Jose State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
056820715
City
San Jose
State
CA
Country
United States
Zip Code
95112
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Shalan, Hadil; Colbert, Alexander; Nguyen, Thanh Truc et al. (2017) Correlating the para-Substituent Effects on Ru(II)-Polypyridine Photophysical Properties and on the Corresponding Hybrid P450 BM3 Enzymes Photocatalytic Activity. Inorg Chem 56:6558-6564
Lam, Quan; Kato, Mallory; Cheruzel, Lionel (2016) Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis. Biochim Biophys Acta 1857:589-597
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
Dwaraknath, Sudharsan; Tran, Ngoc-Han; Dao, Thanh et al. (2014) A facile and versatile methodology for cysteine specific labeling of proteins with octahedral polypyridyl d? metal complexes. J Inorg Biochem 136:154-60
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
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