Human cytochrome p450 family 4 enzymes: function, genetic variation and regulation.

The microsomal cytochrome P450 (CYP) family 4 monooxygenases are the major fatty acid omega-hydroxylases. These enzymes remove excess free fatty acids to prevent lipotoxicity, catabolize leukotrienes and prostanoids, and also produce bioactive metabolites from arachidonic acid omega-hydroxylation. In addition to endogenous substrates, recent evidence indicates that CYP4 monooxygenases can also metabolize xenobiotics, including therapeutic drugs. This review focuses on human CYP4 enzymes and updates current knowledge concerning catalytic activity profiles, genetic variation and regulation of expression. Comparative differences between the human and rodent CYP4 enzymes regarding catalytic function and conditional expression are also discussed.

Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2.

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.

Regulation of human cytochrome P450 4F2 expression by sterol regulatory element-binding protein and lovastatin.

This report provides the first evidence that human P450 4F2 (CYP4F2) is induced by statins, which are widely used to treat hypercholesterolemia. Real time PCR and immunoblots indicate that lovastatin treatment increases expression of the endogenous CYP4F2 gene in human primary hepatocytes and HepG2 cells. The effects of lovastatin on gene expression are often mediated through sterol regulatory element-binding proteins (SREBPs). Immunoblots indicate that lovastatin-treated human hepatocytes display increased proteolytic processing of SREBP-2. In HepG2 cells, co-administration of a potent suppressor of SREBP-2 activation, 25-hydroxycholesterol, inhibits CYP4F2 mRNA induction by lovastatin. HepG2 cells transfected with an expression vector for the active nuclear form of SREBP-1a (nSREBP-1a) also display elevated endogenous CYP4F2 expression. Luciferase reporters containing the CYP4F2 proximal promoter are transactivated by nSREBPs (-1a, -1c, and -2) or a dominant positive form of the SREBP cleavage-activating protein (SCAP), which facilitates activation of endogenous SREBPs. Lovastatin-induced reporter expression is inhibited by overexpressed Insig-1, which prevents proteolytic activation of endogenous SREBPs. Electrophoretic mobility shift assays with in vitro translated nSREBP-1a identified two SREBP binding sites at -169/-152 and -109/-92, relative to the CYP4F2 transcription start site. Mutations in each site abolish SREBP binding. Chromatin immunoprecipitation experiments indicate that more SREBP-1 is associated with the CYP4F2 promoter after overexpression of nSREBP-1a. Transfection studies and mutagenesis indicate that the -109/-92 region is the primary site responsible for the effects of statins. Collectively, these results demonstrate that SREBPs transactivate CYP4F2 transcription and that CYP4F2 induction by statins is mediated by SREBP-2.

Structures of human microsomal cytochrome P450 2A6 complexed with coumarin and methoxsalen.

Human microsomal cytochrome P450 2A6 (CYP2A6) contributes extensively to nicotine detoxication but also activates tobacco-specific procarcinogens to mutagenic products. The CYP2A6 structure shows a compact, hydrophobic active site with one hydrogen bond donor, Asn297, that orients coumarin for regioselective oxidation. The inhibitor methoxsalen effectively fills the active site cavity without substantially perturbing the structure. The structure should aid the design of inhibitors to reduce smoking and tobacco-related cancers.

Conditional regulation of the human CYP4X1 and CYP4Z1 genes.

Cytochrome P450 genes (CYPs) encoding two new subfamilies designated CYP4X1 and CYP4Z1 were identified in the human genome and the Expressed Sequence Tags database. Partial cDNAs encoding both P450s were isolated from human kidney and used to determine tissue distribution. CYP4X1 was predominantly expressed in trachea and aorta, whereas CYP4Z1 mRNA was preferentially expressed in mammary tissue. In T47-D cells, CYP4Z1 mRNA levels were induced by dexamethasone (14-fold) or by progesterone (10-fold). The induction by these compounds was suppressed by co-treatment with the progesterone and glucocorticoid receptor antagonist mifepristone (RU486). In the progesterone receptor negative MCF-7 cells, CYP4Z1 mRNA was induced by dexamethasone but not by progesterone treatment. CYP4Z1 mRNA levels were unaffected by 17beta-estradiol. In confluent cultures of human hepatoma HepG2 cells that stably express a mouse peroxisome proliferator activated receptor-alpha (PPARalpha) mutant, CYP4X1 mRNA was undetectable in vehicle-treated cells but was readily detectable following addition of the PPARalpha agonist Wy14643. This suggests that PPARalpha activation can affect human CYP4X1 gene transcription. These results demonstrate selective tissue expression and implicate PPARalpha in CYP4X1 regulation, and the glucocorticoid and progesterone receptors in CYP4Z1 gene activation.

The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-A resolution.

The structure of P450 3A4 was determined by x-ray crystallography to 2.05-A resolution. P450 3A4 catalyzes the metabolic clearance of a large number of clinically used drugs, and a number of adverse drug-drug interactions reflect the inhibition or induction of the enzyme. P450 3A4 exhibits a relatively large substrate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporin, statins, taxanes, and macrolide antibiotics. Family 3A P450s also exhibit unusual kinetic characteristics that suggest simultaneous occupancy by smaller substrates. Although the active site volume is similar to that of P450 2C8 (PDB code: 1PQ2), the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity. Compared with P450 2C8, the active site cavity of 3A4 is much larger near the heme iron. The lower constraints on the motions of small substrates near the site of oxygen activation may diminish the efficiency of substrate oxidation, which may, in turn, be improved by space restrictions imposed by the presence of a second substrate molecule. The structure of P450 3A4 should facilitate a better understanding of the substrate selectivity of the enzyme.

The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-A resolution.

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 A by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.

Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid binding site.

A 2.7-Angstrom molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the beta1 sheet and helix F' that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F', G', and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism.

Regulation of P450 4A expression by peroxisome proliferator activated receptors.

The induction of P450 4A enzymes by peroxisome proliferators (PPs) and fatty acids is mediated by the peroxisome proliferator activated receptor alpha (PPAR alpha) that binds to response elements in target genes as a heterodimer with the retinoid X receptor (RXR). The consensus sequence recognized by PPAR/RXR heterodimers, contains an imperfect direct repeat of two nuclear receptor binding motifs separated by a single nucleotide. This repeat is preceded by a conserved A/T rich sequence that is required for function. In mice, chronic exposure to PPs results in PPAR alpha mediated liver hypertrophy, hyperplasia and carcinogenesis accompanied by a proliferation of peroxisomes. In contrast, humans exhibit a reduced sensitivity to PP pathogenesis. This could reflect >10-fold lower PPAR alpha levels relative to mice as well as differences in targeted genes. In order to identify PPAR responsive human genes, the human hepatoma cell line, HepG2, was engineered to express increased levels of PPAR alpha. Several genes encoding rate-limiting enzymes and branch points in ketone body formation are regulated by PPAR alpha in these cells. In contrast, significant induction by PP is not evident for peroxisomal fatty acid oxidation that is associated with peroxisome proliferation in mice. Human P450 4A11 is not expressed in dividing cultures of cells with enhanced PPAR alpha levels, but it is expressed in confluent cultures expressing elevated amounts of PPAR alpha.