News and Publications

Cytochrome P-450: Regulation, Structure, and Function

E.F. Johnson, J. Cosme, K.J. Griffin, M.-H. Hsu, F. Jung, W. Song, U. Savas, C. Yang

The microsomal cytochrome P-450 monooxygenases catalyze the hydroxylation of a diverse array of lipophilic compounds, including hormones, carcinogens, and drugs. This metabolic transformation often provides a defensive function whereby toxic compounds can be converted to less toxic forms that are more easily excreted from the body. A relative large group of P-450 enzymes participate in the metabolism of drugs. Genetic differences between individuals in the efficiency or expression of an enzyme can limit bioavailability of a drug because of efficient metabolism or allow accumulation of toxic concentrations because of diminished metabolism. We are working toward an understanding of how the genetic diversity and regulation of these enzymes contribute to a person's ability to avoid the adverse effects of environmental chemicals.

PREDICTING P-450 SUBSTRATE SPECIFICITIES

The therapeutic ramifications of interindividual variation in drug metabolism underscore the need to determine substrate characteristics that would be predictive of the enzymes likely to metabolize a particular drug. To date, the only experimentally determined structures are for soluble bacterial P-450s. Earlier work in our laboratory indicated that genetic variation leading to alterations in the substrate specificity of mammalian P-450s fits a framework model in which key residues function as the principal determinants of the active-site geometries. These empirically defined, key residues often correspond by sequence alignments with substrate-contacting residues in bacterial P-450 structures. More explicit models of the mammalian P-450s incorporate conserved features of the protein folding shared by known P-450 structures to yield 3-dimensional models. We are validating and refining these models by using site-directed mutagenesis designed to predictably alter substrate specificity.

The basis for modeling mammalian P-450s would be greatly improved by a structural determination. However, as is the case for most membrane proteins, diffraction quality crystals of microsomal P-450s have not been produced. Microsomal P-450s contain a single transmembrane segment at the amino terminus. However, deletion of this region does not prevent a peripheral association with membranes when the truncated enzyme is expressed in Escherichia coli. Results of homology modeling suggest that the remaining membrane-binding site most likely does not span the lipid bilayer. Examination of our models of microsomal P-450s suggested probable membrane-binding domains, and mutagenesis of these regions led to the successful expression of conditionally soluble enzymes that have catalytic properties similar to those of the native enzymes. Crystallization trials with these modified P-450s are in progress.

ENZYME INDUCTION

Expression of individual P-450s can increase dramatically after exposure to either drugs or toxic chemicals. Exposure often increases gene transcription, leading to an increase in the concentration of the enzyme and often to an increased capacity to metabolize and detoxify the inducing agent.

Peroxisome proliferators are a large class of foreign compounds that can induce a variety of peroxisomal enzymes and some microsomal P-450s, including the P-450s of subfamily 4A that catalyze hydroxylation of the terminal carbon of fatty acids. The product of this reaction can be oxidized further to a dicarboxylic acid that is metabolized in peroxisomes to shorter, more readily excreted fatty acids. Dicarboxylic acids may in turn signal the cell to increase the number of peroxisomes. This process burns off the caloric content of the fats less efficiently than do other metabolic pathways.

Work in our laboratory indicates that the induction of the P-450 gene 4A6 is mediated by the peroxisome proliferator activated receptor (PPAR). Multiple responsive elements in the promoter and 5´ flanking region of 4A6 are involved. PPAR binds as a heterodimer with the retinoid X receptor (RXR). Characterization of the peroxisome proliferator response elements (PPREs) in 4A6 indicated that they include imperfect direct repeats of the sequence AGGTCA that are also recognized by homodimers of other nuclear receptors such as RXR, apolipoprotein regulatory protein-1, and hepatocyte nuclear factor 4.

Our studies indicate that PPREs also contain an extended binding site that is not required by these other nuclear receptors. However, the extended binding site is similar to that recognized by a different class of nuclear receptors that can function as monomeric transcription factors. We found that PPAR has a second domain adjacent to the zinc finger DNA-binding domain that recognizes the extended sequence. This extended binding site may compensate for imperfections of the direct repeat motif and provide greater specificity for the binding of PPAR/RXR heterodimers.

Long-term exposure to peroxisome proliferators increases the number of hepatic peroxisomes and causes tumorigenesis in sensitive species. PPAR is required for these changes. Several peroxisome proliferators are used to lower serum triglyceride levels in humans, and these beneficial effects are also mediated by PPAR. No evidence suggests that these hyperlipidemic drugs produce peroxisome proliferation or tumors in humans. Our research indicates that expression of hepatic PPAR is lower in humans than in sensitive species. This difference is due, in part, to significant nonproductive processing of the PPAR RNA transcript in humans. The low abundance of human PPAR may be sufficient to mediate moderate responses of beneficial target genes to hyperlipidemic drugs. In contrast, the relatively high abundance of PPAR in sensitive species may lead to inappropriate regulation of additional genes because of greater competition with other regulatory factors at marginal response elements.

Activators of PPARs are important in governing the balance between fat storage, glucose production from fats, and destruction of excess fats. A number of endogenous fatty acids appear to be ligands for PPARs but are generally weaker agonists than are oxidized metabolites of unsaturated fatty acids. PPAR is often expressed in tissues that express P-450s that catalyze the formation of epoxides from unsaturated fatty acids. These epoxides are rapidly metabolized to dihydroxylated compounds. Our studies indicate that dihydroxylated compounds formed from arachidonic acid are selective agonists of the different PPAR isoforms, depending on the site of epoxidation. These agonists have relatively high affinities for the receptor, compared with the affinities of other peroxisome proliferators and other fatty acids, and thus may play a role in the signal transduction that regulates PPAR activity.

PUBLICATIONS

Dierks, E.A., Zhang, Z., Johnson, E.F., Ortiz de Montellano, P.R. The catalytic site of cytochrome P-450 4A11 (CYP4A11) and its L131 mutant. J. Biol. Chem. 273:23055, 1998.

Guengerich, F.P., Parikh, A., Johnson, E.F., Richardson, T.H., von Wachenfeldt, C., Cosme, J., Jung, F., Strassburg, C.P., Manns, M.P., Tukey, R.H., Pritchard, M., Fournel-Gigeux, S., Burchell, B. Heterologous expression of human drug-metabolizing enzymes. Drug Metab. Dispos. 25:1234, 1997.

Hsu, M.-H., Palmer, C.N.A., Song, W., Griffin, K.J., Johnson, E.F. A carboxy terminal extension of the zinc finger domain contributes to the specificity and polarity of peroxisome proliferator activated receptor DNA binding. J. Biol. Chem., in press.

Palmer, C.N.A., Hsu, M.-H., Griffin, K.J., Raucy, J.L., Johnson, E.F. Peroxisome proliferator activated receptor expression in human liver. Mol. Pharmacol. 53:14, 1998.