The structure of P450 3A4 was determined by x-ray crystallography to 2.05-Å 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.Determination of the structure of P450 1 3A4 is of particular interest because the enzyme contributes extensively to human drug metabolism due to its high level of expression in liver (1) and broad capacity to oxidize structurally diverse substrates (2, 3). The enzyme also provides a significant barrier to the bioavailability of new drug candidates contributing to attrition from the developmental pipeline. Additionally, metabolic drug-drug interactions between substrates and inhibitors of the enzyme can profoundly affect the safety or efficacy of drug therapy (4, 5).Our laboratory was the first to demonstrate that microsomal P450s could be crystallized for structural determination by x-ray crystallography when the proteins were modified for expression as conditional membrane proteins (6, 7). As a result, structures for P450s in family 2, subfamilies B and C are now available (8 -14). P450s of family 3, subfamily A exhibit less than 40% amino acid sequence identity with family 2 P450s. In addition, family 3 P450s often exhibit complex kinetic properties such as substrate and effector activation. Effectors or alternative substrates can modulate the apparent binding affinity for other inhibitors (15) and substrates (16). Moreover, there are a number of examples where alternative substrates fail to inhibit the oxidation of specific substrates leading to kinetic models based on the occupancy of the substrate-binding cavity by two substrates that each can be oxidized by the reactive, hypervalent oxy-perferryl heme intermediate without interference from the other (17, 18). The observation that P450 3A4 oxidizes some of the largest substrates identified for P450s, such as cyclosporin, bromocryptine, and macrolide antibiotics (3), has generally suggested the likelihood that...

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å 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.P450 2C9 is one of three human microsomal cytochrome P450s (CYPs) 1 in subfamily 2C that contribute extensively to the hepatic metabolism of therapeutic drugs. The P450 2C9 locus is polymorphic leading to a diminished capacity to clear specific drugs in genetically affected individuals. For P450 2C9 substrates, such as warfarin or phenytoin, that have low therapeutic margins of safety, diminished metabolic capacity because of genetic polymorphisms or drug-drug interactions can lead to toxicity at normal therapeutic doses (1). P450 2C9 has also been implicated in the synthesis of arachidonic acid epoxides in extrahepatic tissues where they regulate blood pressure (2). Like other P450 subfamilies, the 2C enzymes share roughly 70% or greater amino acid identity. However, the 2C genes have duplicated and diverged rapidly as mammalian species evolved, leading to different numbers of enzymes in various species and highly divergent substrate selectivities. This diversity reflects high rates of non-synonymous substitutions that often alter residues that line the active site cavity and determine substrate selectivity.Human P450s 2C9 and 2C19 are closely related with roughly 91% amino acid identity. Although they exhibit distinct substrate selectivities, residues predicted to line the active site cavity, based on the published structures of other mammalian P450s (3-6), do not differ between the two enzymes. This suggests that conformation changes are likely to underlie differences in the substrate selectivities of P450s 2C9 and 2C19 and that the structure(s) of one or both will differ from those published previously. This is supported by studies of chimeric enzymes generated from P450s 2C9 and 2C19 that have generally identified amino acid residues that are predicted to reside outside the substrate binding cavity as determinants of their distinct catalytic properties (7-9). P450 2C9 exhibits a selectivity for the oxidation of relatively small, lipophilic anions such as the non-steroidal anti-inflammator...

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 ␣-naphthoflavone, determined to a resolution of 1.95 Å . ␣-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. Enzymes of the cytochrome P450 (CYP)5 superfamily play a significant physiologic role in the detoxication of foreign compounds and the biosynthesis of endogenous compounds, including steroid hormones, bile acids, and cholesterol. The enzymes comprising P450 families 1, 2, and 3 contribute most extensively to the biotransformation of xenobiotics to more polar metabolites that are more readily excreted. In humans and most mammals, family 1 contains three well characterized P450 monooxygenases; 1A1, 1A2, and 1B1. These enzymes are generally distinguished from P450s in other families by their capacity to oxidize a variety of polynuclear aromatic hydrocarbons (PAHs).6 Moreover, the expression levels of the three enzymes are induced by exposure to PAHs (1). The induction is mediated by a ligand-activated transcription factor, the aryl hydrocarbon receptor, which is a basic-loop-helix PAS domain protein that binds to enhancer elements flanking the CYP1A1, CYP1A2, and CYP1B1 genes and stimulates transcription.The oxidation of PAHs is generally protective. However, some P450-catalyzed reactions can transform these relatively inert compounds into genotoxic metabolites that can initiate mutagenesis and cancer. Human P450 1A2 is notable among family 1 enzymes for the capacity to N-oxidize arylamines, the major metabolic process in the bioactivation of arylamines to potent mutagenic or carcinogenic compounds (2). ␣-Naphthoflavone (ANF), a prototype flavonoid, is known to competitively inhibit P450s of family 1, albeit at different concentrations, and has been used to discriminate between P450 family 1 enzymes (3). Flavonoids have gained recent interest in vi...

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.

The peroxisome proliferator activated receptor ␣ (PPAR) is a member of the steroid/hormone receptor superfamily that mediates the peroxisome proliferator-dependent transcriptional activation of genes encoding several peroxisomal and microsomal enzymes as well as peroxisome proliferation. Human liver is refractory to the pathological effects of peroxisome proliferators that are seen in mice. With the use of RNase protection assays, the ratio of hepatic PPAR␣ mRNA to ␤-actin mRNA was found to be 1 order of magnitude lower in humans than that observed in mice. In addition, the isolation of human cDNA for PPAR␣ that does not encode a functional PPAR because it lacks exon 6 as a result of alternate RNA splicing suggested that this process might also diminish the expression of PPAR␣. RNase protection analysis of total RNA revealed the presence of splice variants lacking exon 6 at significant levels in all 10 human liver samples examined. Supershift analysis using the CYP4A6-Z peroxisome proliferator response element and antisera specific for PPAR␣ revealed easily detectable amounts of PPAR␣ DNA binding activity in mouse liver lysates, whereas human liver lysates contained Ͼ10-fold lower amounts of PPAR␣ DNA binding activity. In contrast to mouse lysates, the amount of PPAR␣ binding in human lysates was generally less than that of other unidentified proteins. These results suggest that although humans retain the coding potential for a functional receptor, the low levels of PPAR␣ expression in liver may be insufficient to compete effectively with other proteins that bind to peroxisome proliferator response elements.