The cytochrome P450 (cyt P450) superfamily consists of more than 11,000 members. They are ubiquitous, being found in all kingdoms of living organisms and plants and are referred to as Mother Nature's blowtorch, due to their ability to oxidize a vast number of stable chemical entities. Humans possess 56 different cyts P450, many of which are essential for early development and life itself. Other human cyts P450 determine the toxicity, duration of action, and elimination of the vast majority of therapeutic agents, carcinogens, and environmental agents to which humans are exposed. Xenobiotic metabolizing cyts P450 are also responsible for the majority of drug-drug interactions and adverse drug reactions. A third group of cyts P450 are responsible for the biosynthesis or metabolism of essential endogenous compounds. This includes virtually all steroids (cholesterol, bile acids, estrogens, testosterone, cortisol, and vitamin D) and many lipids and eicosanoids. Cyts P450 exists in virtually every organ and tissue of humans. The cyts P450 are not self-sufficient but rather require interactions with other proteins in order to function. Cyt P450 reductase and cytochrome b5 (cyt b5), which provide electrons to cyt P450, are two proteins that support the activity of cyt P450. The long-term goal of this project is to understand the structural and mechanistic basis for the regulation of the activity of the membrane-bound microsomal cyts P450 by its redox partners, cyt P450 reductase and cyt b5. The short-term goals of this proposal, using both human and model cyts P450, are to understand the biochemical basis of how the redox partners of cyt P450 regulate its activity, substrate specificity, and catalytic mechanism. Experimental techniques, including site-directed mutagenesis, rapid quenching of cyt P450 activity by chemical means, and freezing, HPLC-mass spectrometry, and quantum mechanical/molecular mechanical calculations will be employed to elucidate how the activity of microsomal cyts P450 is regulated by its redox partners. Understanding how nature designs cyt P450 active sites is a fundamental question with implications for predicting and eventually modifying the routes of metabolism of a large number of environmental contaminants such as phthalates, bisphenol A, polychlorinated biphenyls (PCBs) and many currently used drugs, including chemotherapeutic agents, psychoactive compounds, and cardiovascular therapies. Knowledge of the molecular mechanism by which the activity of human cyts P450 can be regulated will also prove to be a tremendous asset in developing drugs and procedures to alter the large number of critical physiologic processes in which the human cyts P450 participate, as well as in designing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents and environmental contaminants.

Public Health Relevance

The proposed investigation of how the human enzymes responsible for drug metabolism, drug-drug interactions, blood clotting, and cholesterol levels function at a molecular level will provide new information that will prove to be an asset 1) in developing novel procedures to alter the large number of physiologic processes in which the 56 human cyts P450 participate and 2) in designing and synthesizing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents, and """"""""greener"""""""" environmental contaminants.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM094209-01A1
Application #
8108965
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Anderson, Vernon
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
1
Fiscal Year
2011
Total Cost
$413,980
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Yang, Yuting; Bu, Weishu; Im, Sangchoul et al. (2018) Structure of cytochrome P450 2B4 with an acetate ligand and an active site hydrogen bond network similar to oxyferrous P450cam. J Inorg Biochem 185:17-25
Xia, Chuanwu; Rwere, Freeborn; Im, Sangchoul et al. (2018) Structural and Kinetic Studies of Asp632 Mutants and Fully Reduced NADPH-Cytochrome P450 Oxidoreductase Define the Role of Asp632 Loop Dynamics in the Control of NADPH Binding and Hydride Transfer. Biochemistry 57:945-962
Rwere, Freeborn; Xia, Chuanwu; Im, Sangchoul et al. (2016) Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone. J Biol Chem 291:14639-61
Peng, Hwei-Ming; Im, Sang-Choul; Pearl, Naw May et al. (2016) Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production. Biochemistry 55:4356-65
Davydov, Roman; Im, Sangchoul; Shanmugam, Muralidharan et al. (2016) Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy. Biochemistry 55:869-83
Huang, Rui; Zhang, Meng; Rwere, Freeborn et al. (2015) Kinetic and structural characterization of the interaction between the FMN binding domain of cytochrome P450 reductase and cytochrome c. J Biol Chem 290:4843-55
Yang, Yuting; Zhang, Haoming; Usharani, Dandamudi et al. (2014) Structural and functional characterization of a cytochrome P450 2B4 F429H mutant with an axial thiolate-histidine hydrogen bond. Biochemistry 53:5080-91
Vivekanandan, Subramanian; Ahuja, Shivani; Im, Sang-Choul et al. (2014) ¹H, ¹³C and ¹?N resonance assignments for the full-length mammalian cytochrome b? in a membrane environment. Biomol NMR Assign 8:409-13
Huang, Rui; Yamamoto, Kazutoshi; Zhang, Meng et al. (2014) Probing the transmembrane structure and dynamics of microsomal NADPH-cytochrome P450 oxidoreductase by solid-state NMR. Biophys J 106:2126-33
Pandey, Manoj Kumar; Vivekanandan, Subramanian; Ahuja, Shivani et al. (2013) Cytochrome-P450-cytochrome-b5 interaction in a membrane environment changes 15N chemical shift anisotropy tensors. J Phys Chem B 117:13851-60

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