Scientific Report 2005

Molecular and Experimental Medicine

Division Of Biochemistry

Cytochrome P450: Regulation, Structure, and Function

E.F. Johnson, K.J. Griffin, M.-H. Hsu, Ü. Savas,G.A. Schoch, J.K. Yano

Mammalian genomes generally contain 50–100 genes for the P450 monooxgenases. Some P450s have specialized roles in autocrine, paracrine, and endocrine signal transduction pathways, and most P450s play defensive roles by converting toxic compounds to less toxic forms that are more soluble and more easily excreted than are the parent compounds. Each xenobiotic-metabolizing P450 generally oxidizes structurally diverse substrates, leading to a wide-ranging protective capacity for elimination of toxic chemicals. We wish to understand how the structural diversity and regulation of the P450s that metabolize xenobiotics contribute to a person’s ability to avoid the adverse effects of environmental chemicals and alter the clearance and bioavailability of therapeutic drugs.

Although extensive information on the conditional expression of P450 genes in experimental species is available, in humans, the transcriptional responses of P450 genes to environmental stimuli and to physiologic changes are poorly understood. To address this problem, we used both cell lines and transgenic mice to study expression of human family 4 P450 genes. These genes encode enzymes that are involved in both signal transduction and xenobiotic metabolism. Studies with cell lines are providing new information about endocrine and autocrine signal transduction pathways that govern the conditional expression of these genes in response to nutritional, hormonal, and xenobiotic signals. Research is in progress to determine whether more complex physiologic conditions such as pregnancy or energy restriction alter the expression of the human enzymes in transgenic mice.

We also pioneered using x-ray crystallography to determine the atomic structures of P450s that contribute extensively to drug metabolism in humans. In collaboration with C.D. Stout, Department of Molecular Biology, we are defining the structural features of individual human P450s that contribute to the unique catalytic selectivities of the enzymes. Our goal is to better understand the adverse affects of the oxidation of drugs and toxins and the potential for metabolic drug-drug interactions that can arise from inhibition of P450s if multidrug therapies are used.

Mammalian P450s are tethered to the endoplasmic reticulum by a transmembrane segment at the amino terminus and by additional interactions of the catalytic domain with the cytoplasmic side of the membrane. Although membrane proteins are difficult to crystallize, we developed methods to express, purify, and crystallize genetically modified mammalian P450s. Through these studies, we discovered how the flexibility of the P450s and the diversity of their amino acid sequences shape catalytic specificity.

Specific P450s, such as 3A4 and 2C8, oxidize relatively large compounds such as the immunosuppressant cyclosporin and the antitumor drug paclitaxel. Structures determined for 3A4 and 2C8 indicate how the architecture of these enzymes has adapted to accommodate these large substrates.

P450s can also oxidize relatively small substrates such as chloroform, ethanol, and nicotine. Recently, we solved the structure of the P450 2A6, the principal nicotine-oxidizing enzyme. Although 2A6 plays a prominent role in detoxification of nicotine, it also can activate the tobacco smoke–specific carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the carcinogenic form. Several reports indicate that because of the increased side effects of nicotine, persons who are genetically deficient in 2A6 activity are less likely to smoke than are persons not genetically deficient in this activity, and those who are deficient in 2A6 activity are less likely to have lung cancer if they do smoke. The structure of 2A6 will contribute to the design and development of inhibitors that could alter smoking behavior and diminish the likelihood of tobacco-related lung cancers.

Publications

Mellet, A., Marques-Soares, C., Schoch, G., Macherey, A.-C., Jaouen, M., Dansette, P.M., Sari, M.-A., Johnson, E.F., Mansuy, D. Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants. Biochemistry 43:15379, 2004.

Poulos, T.L., Johnson, E.F. Structures of cytochrome P450 enzymes. In: Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed. Ortiz de Montellano, P.R. (Ed.). Plenum Publishing, New York, 2005, p. 87.

Savas, Ü., Hsu, M.-H., Griffin, K.J., Bell, D.R., Johnson, E.F. Conditional regulation of the human CYP4X1 and CYP4Z1 genes. Arch. Biochem. Biophys. 436:377, 2005.

Wester, M.R., Yano, J.K., Schoch, G.A., Yang, C., Griffin, K.J., Stout, C.D., Johnson, E.F. The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-Å resolution. J. Biol. Chem. 279:35630, 2004.

Yano, J.K., Wester, M.R., Schoch, G.A., Griffin, K.J., Stout, C.D., Johnson, E.F. The structure of human microsomal cytochrome P450 3A4 determined by x-ray crystallography to 2.05-Å resolution. J. Biol. Chem. 279:38091, 2004.