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...

The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-Å crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the ␣-helical and ␤-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetryrelated molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress. T he cytochromes P450 (P450s) are a superfamily of hemecontaining monooxygenases. They are responsible for the metabolism of an unusually wide range of endogenous and exogenous substrates, including synthesis of steroid hormones, bile acids, and cholesterol, and the degradation of steroids, fatty acids, drugs, toxins, and procarcinogens (1). P450s from families 1, 2, and 3 have evolved to convert lipophilic xenobiotics to more polar metabolites readily conjugated by phase II enzymes, and thus targeted for elimination. The stereo-and regiospecificity of metabolite formation by individual xenobiotic metabolizing P450s suggest very specific substrate-enzyme interactions, whereas the range of substrates metabolized suggests an induced fit type of substrate recognition. Understanding the basis for this specific, yet versatile, metabolism by mammalian xenobiotic metabolizing P450s has been limited by the dearth of structural information for these membrane proteins.In 2000, the first mammalian P450 structure was published (2, 3), that of P450 2C5, which was engineered to delete the single N-terminal transmembrane domain and to mutate a peripheral membrane-binding site. Recent structures of 2C5 bound with the substrates, diclofenac (4) and 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ) (5), indicate that flexible regions of the protein adapt for substrate binding, and that ligands may bind in multiple orientations. The 2C5 structures generally reveal the enzyme closed around these substrates wi...

The structure of rabbit microsomal cytochrome P450 2C5/3LVdH complexed with a substrate, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ), was determined by X-ray crystallography to 2.3 A resolution. Substrate docking studies and electron density maps indicate that DMZ binds to the enzyme in two antiparallel orientations of the long axis of the substrate. One orientation places the principal site of hydroxylation, the 4-methyl group, 4.4 A from the heme Fe, whereas the alternate conformation positions the second, infrequent site of hydroxylation at >5.9 A from the heme Fe. Comparison of this structure to that obtained previously for the enzyme indicates that the protein closes around the substrate and prevents open access of water from bulk solvent to the heme Fe. This reflects a approximately 1.5 A movement of the F and G helices relative to helix I. The present structure provides a complete model for the protein from residues 27-488 and defines two new helices F' and G'. The G' helix is likely to contribute to interactions of the enzyme with membranes. The relatively large active site, as compared to the volume occupied by the substrate, and the flexibility of the enzyme are likely to underlie the capacity of drug-metabolizing enzymes to metabolize structurally diverse substrates of different sizes.