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...

The 4-and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes. The 3-hydroxylation step leading to the formation of caffeic acid from p-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygenase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta. In this study, we show that the gene encoding CYP98A3, which was the best possible P450 candidate for a 3-hydroxylase in the Arabidopsis genome, is highly expressed in inflorescence stems and wounded tissues. Recombinant CYP98A3 expressed in yeast did not metabolize free pcoumaric acid or its glucose or CoA esters, p-coumaraldehyde, or p-coumaryl alcohol, but very actively converted the 5-O-shikimate and 5-O-D-quinate esters of trans-p-coumaric acid into the corresponding caffeic acid conjugates. The shikimate ester was converted four times faster than the quinate derivative. Antibodies directed against recombinant CYP98A3 specifically revealed differentiating vascular tissues in stem and root. Taken together, these data show that CYP98A3 catalyzes the synthesis of chlorogenic acid and very likely also the 3-hydroxylation of lignin monomers. This hydroxylation occurs on depsides, the function of which was so far not understood, revealing an additional and unexpected level of networking in lignin biosynthesis.Systematic genome sequencing is revealing a large number of orphan genes and their phylogenetic relatedness to genes with characterized function. EST 1 sequences, on the other hand, are providing preliminary information on levels, patterns of expression, and conservation of genes among species. Taken together, such information can be exploited as a clue to gene function and to track down missing steps in important pathways.The sequencing of the whole genome of Arabidopsis thaliana has revealed 273 cytochrome P450 genes distributed into 45 families and subfamilies (drnelson.utmem.edu/CytochromeP450. html, www.biobase.dk/P450/). P450 proteins thus form the largest superfamily of enzymes involved in plant metabolism, but the function of 80% of these enzymes is still unknown. Our attention was first drawn to the CYP98 family by its phylogeny and structure. An analysis of P450 phylogeny in A. thaliana (Fig. 1) shows that the CYP98 family is most closely related to CYP73A5, coding for the cinnamic-acid 4-hydroxylase, the second enzyme and first P450 in the phenylpropanoid pathway (1). CYP73A5 and the CYP98 family seem to have evolved from the same ancestor as CYP79 enzymes, some of which also, in common with CYP73A5, use aromatic substrates derived from the shikimate pathway (2, 3). It was thus tempting to speculate that the substrate of CYP98 enzymes was derived from aromatic amino acids as well. The Arabidopsis CYP98 family is formed by only three genes. CYP98A3 is present in single copy; CYP98A8 and CYP98A9 are 69% identical to one another and only 52% identical to CYP98A3. All P450 genes in the phenylpropanoid pathway (CYP73A5, CYP84A1, and CYP...

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with G␣ when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of G␣, RGS domain binding consequently accelerates G␣-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domaincontaining proteins with varied G␣ substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential G␣ selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/G␣ complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the G␣ substrate, suggests that unique structural determinants specific to particular RGS protein/G␣ pairings exist and could be used to achieve selective inhibition by small molecules.GTPase-accelerating proteins ͉ NMR structure ͉ RGS proteins ͉ x-ray crystallography G protein-coupled receptors (GPCRs) are critical for many physiological processes including vision, olfaction, neurotransmission, and the actions of many hormones (1). As such, GPCRs are the largest fraction of the ''druggable proteome,'' and their ligand-binding and signaling properties remain of considerable interest to academia and industry (2). GPCRs catalyze activation of heterotrimeric G proteins comprising a guanine nucleotide-binding G␣ subunit and an obligate G␤␥ dimer (3). Receptor-promoted activation of G␣␤␥ causes exchange of GDP for GTP by G␣ and resultant dissociation of G␤␥. GTP-bound G␣ and freed G␤␥ then regulate intracellular effectors such as adenylyl cyclase, phospholipase C, ion channels, RhoGEFs, and phosphodiesterases (1, 4). This ''G protein cycle'' is reset by the intrinsic GTP hydrolysis activity of G␣, producing G␣⅐GDP that favors heterotrimer reformation and, consequently, signal termination. Thus, a major determinant of the duration and magnitude of GPCR signaling is the lifetime of G␣ in the GTP-bound state.Regulators of G protein signaling are GTPase-accelerating proteins (GAPs) for G␣ subunits and thus facilitate GPCR signal termination (5). GAP activity is conferred by an RGS domain present in one or more copies within members of this protein superfamily (5). The archetypal RGS domain is composed of nine ␣-helices (6) and binds most avidly to G␣ in the transi...

Metabolic plasticity, which largely relies on the creation of new genes, is an essential feature of plant adaptation and speciation and has led to the evolution of large gene families. A typical example is provided by the diversification of the cytochrome P450 enzymes in plants. We describe here a retroposition, neofunctionalization, and duplication sequence that, via selective and local amino acid replacement, led to the evolution of a novel phenolic pathway in Brassicaceae. This pathway involves a cascade of six successive hydroxylations by two partially redundant cytochromes P450, leading to the formation of N1,N5-di(hydroxyferuloyl)-N10-sinapoylspermidine, a major pollen constituent and so-far-overlooked player in phenylpropanoid metabolism. This example shows how positive Darwinian selection can favor structured clusters of nonsynonymous substitutions that are needed for the transition of enzymes to new functions.

A 2.7-Å 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 ␤1 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.

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