Cytochrome P450 (P450) enzymes are the major catalysts involved in the metatiolism of carcinogens, drugs, and steroids. Variations in the catalytic activities have a variety of effects in homeostasis and clinical practice. Continued studies on human P450s are proposed, with a focus on molecular understanding of function. (1) Of the 57 human P450 genes, 13 still have limited if any knowledge regarding function. We propose to establish sites of mRNA expression, express these """"""""orphan"""""""" P450s in heterologous systems, and examine their abilities to activate a wide variety of chemical carcinogens. In addition, several HPLC-mass spectrometry approaches will be used for identification of products and substrates, with tissue extracts as sources of substrates and the P4SOs as reagents. This part of the project is an effort towards understanding the functional genomics of human P450s. A related aspect is establishment of the roles of individual human P450s in morphine biosynthesis, forwhich strong evidence has been recently presented by others. (2) Comparisons of the Idnetics of human P450s already studied in detail (1A2, 2A6, 2D6,2E1, 3A4) will be done with several other P450s reported to have much higher rates of catalysis, with the goal of understanding which steps limit the (human P4S0) reactions. These studies will involve a variety of steady-state, pre-steady-state, and isotope effect approaches. (3) Kinetic analysis of multi-reaction P450s will be done, including P450S 51Al (ianosterol 14a-demethylation), 19A1 (aromatase, oxidation of testosterone to 17p-estradiol), and 2A6 (oxidation of indoles), wrth a goal of defining the processivity of these systems. Several pre-steady-state and analysis approaches can be readily applied to the problem, with the goal of understanding the release of intermediates. (4) P450S 3A4 and 2A6 will be analyzed regarding hypotheses about cooperativity and induced fit in substrate binding and catalysis. The P450 3A4 work will focus on pre-steady-state i^inetics of substrate binding, along with thermodynamic analysis of binding. The work on P4S0 2A6 induced fit will involve collaborative work on X-ray crystallography of mutants that have demonstrated expansion of the active site. These results should reveal whether a P450 has a fixed structure or can """"""""adapt"""""""" to individual substrates, Collectively, these studies have the goal of providing more understanding of the roles of human P450s in oxidation of drugs, carcinogens, and endogenous compounds and their potential contributions to cancer and other diseases and roles in disease treatments.

Public Health Relevance

The P450 enzymes are the main catalysts involved in the metabolism of carcinogens and daigs, and the balance between bioactivation (to dangerous products) and detoxication is important in health issues and also varies among individuals. Many of the biochemical properties of some human P450s are pooriy understood, and several of these enzymes are postulated to have important roles in chemical carcinogenesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA090426-13
Application #
8471066
Study Section
Special Emphasis Panel (NSS)
Program Officer
Johnson, Ronald L
Project Start
2001-05-20
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
13
Fiscal Year
2013
Total Cost
$342,100
Indirect Cost
$122,805
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Kramlinger, Valerie M; Nagy, Leslie D; Fujiwara, Rina et al. (2016) Human cytochrome P450 27C1 catalyzes 3,4-desaturation of retinoids. FEBS Lett 590:1304-12
Ačimovič, Jure; Goyal, Sandeep; Košir, Rok et al. (2016) Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis. Sci Rep 6:28462
Yoshimoto, Francis K; Gonzalez, Eric; Auchus, Richard J et al. (2016) Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN. J Biol Chem 291:17143-64
Shimada, Tsutomu; Takenaka, Shigeo; Murayama, Norie et al. (2016) Oxidation of pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene by human cytochrome P450 2A13. Xenobiotica 46:211-24
Enright, Jennifer M; Toomey, Matthew B; Sato, Shin-ya et al. (2015) Cyp27c1 Red-Shifts the Spectral Sensitivity of Photoreceptors by Converting Vitamin A1 into A2. Curr Biol 25:3048-57
Rendic, Slobodan; Guengerich, F Peter (2015) Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals. Chem Res Toxicol 28:38-42
Kramlinger, Valerie M; Alvarado Rojas, Mónica; Kanamori, Tatsuyuki et al. (2015) Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine, a Key Step in Endogenous Mammalian Morphine Biosynthesis. J Biol Chem 290:20200-10
Bojić, Mirza; Sedgeman, Carl A; Nagy, Leslie D et al. (2015) Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450. Eur J Pharm Sci 73:49-56
Shimada, Tsutomu; Takenaka, Shigeo; Murayama, Norie et al. (2015) Oxidation of Acenaphthene and Acenaphthylene by Human Cytochrome P450 Enzymes. Chem Res Toxicol 28:268-78
Guengerich, F Peter (2015) Introduction: Metals in Biology: METALS AT THE HOST-PATHOGEN INTERFACE. J Biol Chem 290:18943-4

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