Flexibility and stability of the structure of cytochromes P450 3A4 and BM-3.

The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun. 252, 166-172].     

Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 ?), CYP2A13 (3.0 ?), CYP2E1 (2.35 ?), and the CYP2A6 mutant enzyme, CYP2A6?I208S/I300F/G301A/S369G (2.1 ?) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.     

Different binding modes of two flaviolin substrate molecules in cytochrome P450 158A1 (CYP158A1) compared to CYP158A2

Cytochrome P450 158A2 (CYP158A2) has been shown to catalyze an unusual oxidative C-C coupling reaction to polymerize flaviolin and form highly conjugated pigments (three isomers of biflaviolin and one triflaviolin) in Streptomyces coelicolor A3(2) which protect the soil bacterium from deleterious effects of UV irradiation (Zhao B. et al. (2005) J. Biol. Chem. 280, 11599-11607). The present studies demonstrate that the subfamily partner CYP158A1, sharing 61% amino acid identity with CYP158A2, can also catalyze the same flaviolin dimerization reactions, but it generates just two of the three isomers of biflaviolin that CYP158A2 produces. Furthermore, the two CYP158A1 products have very different molar ratios compared with the corresponding CYP158A2 products, indicating that each enzyme maintains its own stereo- and regiospecificity. To find an explanation for these differences, three CYP158A1 structures have been solved by X-ray crystallography and have been compared with those for CYP158A2. The structures reveal surprising differences. Particularly, only one flaviolin molecule is present close to the heme iron in CYP158A1, and the second flaviolin molecule binds at the entrance of the putative substrate access channel on the protein distal surface 9 A away. Our work describes two members of the same P450 subfamily, which produce the same products by oxidative C-C coupling yet show very different structural orientations of substrate molecules in the active site.     

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.     

Homology modeling and substrate binding study of human CYP4A11 enzyme

Although both bacterial CYP102 (P450BM3) and mammalian CYP4A isozymes share a common function as fatty acid hydroxylases, distinctly different preferred sites of oxidation are observed with the CYP102 performing the usual non-terminal hydroxylation or epoxidation and the CYP4A enzymes performing the unusual and enigmatic terminal hydroxylation. The origin of this unique product specificity in human CYP4A11 has been explored in this work, focusing on possible differences in the binding site architecture of the two isozymes as the cause. To this end, 3D model structures of the human CYP4A11 enzyme were built and compared to the X-ray structure of CYP102. The substrate-binding channel identified in CYP4A11 was found to have a much more sterically restricted active site than that in CYP102 that could cause limited access of long-chain fatty acid to the ferryl oxygen leading to the preferred omega-hydroxylation. Results of docking of a common substrate, lauric acid, into the binding site of both CYP4A11 and CYP102 and molecular dynamics simulations provided additional support for this hypothesis. Specifically, in the CYP4A11-lauric acid simulations, the omega hydrogens were closest to the ferryl oxygen most of the time. By contrast, in the CYP102-lauric acid complex, the substrate could penetrate further into the active site providing access of the non-terminal (omega-1, omega-2) positions to the ferryl oxygen. These results, taken together, have elucidated the origin of the unusual product specificity of CYP4A11 and illustrated the central role of binding site architecture in subtle modulation of function.     

Structure of the human lung cytochrome P450 2A13

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.     

Active sites of two orthologous cytochromes P450 2E1: differences revealed by spectroscopic methods

Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.     

A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.     

Structural Basis of Drug Binding to CYP46A1, an Enzyme That Controls Cholesterol Turnover in the Brain

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.     

Homology model of 1alpha,25-dihydroxyvitamin D3 24-hydroxylase cytochrome P450 24A1 (CYP24A1): active site architecture and ligand binding

Homology models of cytochrome P450 24A1 (CYP24A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using molecular operating environment (MOE) software the lowest energy CYP24A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP24A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP24A1 model. The natural substrate, 1,25-dihydroxyvitamin D(3) (calcitriol) and the CYP24 inhibitor (R)-N-(2-(1H-imidazol-1-yl)-2-phenylethyl)-4'-chlorobiphenyl-4-carboxamide ((R)-VID-400) were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure.     

Structures of cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) catalyzes the initial step in the clearance of many pharmaceuticals and foreign chemicals. The structurally diverse nature of CYP3A4 substrates complicates rational prediction of their metabolism and identification of potential drug interactions. The first molecular structures of human CYP3A4 were recently determined, revealing an active site of sufficient size and topography to accommodate either large ligands or multiple smaller ligands, as suggested by the heterotropic and homotropic cooperativity of the enzyme.     

Covalently linked heme in cytochrome p4504a fatty acid hydroxylases

Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes. Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli. However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression. The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric. In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site. This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group.     

Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.     

Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.     

A conformationally restricted uniconazole analogue as a specific inhibitor of rice ent-kaurene oxidase, CYP701A6

The plant growth retardant uniconazole (UNI), which has been used as an effective inhibitor of ent-kaurene oxidase (CYP701A) involved in gibberellin biosynthesis, also strongly inhibits ABA 8'-hydroxylase (CYP707A), a key enzyme in abscisic acid catabolism. Azole P450 inhibitors bind to the P450 active site by both coordinating to the heme-iron atom via an sp(2) nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from its small molecular size and flexible conformation that allows it to fit into active sites differing in size and shape. To find a selective inhibitor of CYP701A based on this hypothesis, we examined inhibitory activities of three types of UNI analogues, which were conformationally constrained, enlarged in width, and enlarged in length, against recombinant rice CYP701A6 and Arabidopsis CYP707A3. Conformationally restricted analogues, UFAP2 and UFAP2N, inhibited CYP701A6 as strongly as UNI, whereas it inhibited CYP707A3 less than UNI.     

Time-resolved fluorescence studies of heterotropic ligand binding to cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) is a major enzymatic determinant of drug and xenobiotic metabolism that demonstrates remarkable substrate diversity and complex kinetic properties. The complex kinetics may result, in some cases, from multiple binding of ligands within the large active site or from an effector molecule acting at a distal allosteric site. Here, the fluorescent probe TNS (2-p-toluidinylnaphthalene-6-sulfonic acid) was characterized as an active site fluorescent ligand. UV-vis difference spectroscopy revealed a TNS-induced low-spin heme absorbance spectrum with an apparent K(d) of 25.4 +/- 2 microM. Catalytic turnover using 7-benzyloxyquinoline (7-BQ) as a substrate demonstrated TNS-dependent inhibition with an IC(50) of 9.9 +/- 0.1 microM. These results suggest that TNS binds in the CYP3A4 active site. The steady-state fluorescence of TNS increased upon binding to CYP3A4, and fluorescence titrations yielded a K(d) of 22.8 +/- 1 microM. Time-resolved frequency-domain measurement of TNS fluorescence lifetimes indicates a testosterone (TST)-dependent decrease in the excited-state lifetime of TNS, concomitant with a decrease in the steady-state fluorescence intensity. In contrast, the substrate erythromycin (ERY) had no effect on TNS lifetime, while it decreased the steady-state fluorescence intensity. Together, the results suggest that TNS binds in the active site of CYP3A4, while the first equivalent of TST binds at a distant allosteric effector site. Furthermore, the results are the first to indicate that TST bound to the effector site can modulate the environment of the heterotropic ligand.     

Enantioselectivity of inhibition of cytochrome P450 3A4 (CYP3A4) by ketoconazole: Testosterone and methadone as substrates

Racemic ketoconazole (KTZ) was the first orally active azole antifungal agent used in clinical practice and has become widely used in the treatment of mucosal fungal infections associated with AIDS immunosuppression and cancer chemotherapy. However, the use of KTZ has been limited because of adverse drug-drug interactions. KTZ blocks ergosterol biosynthesis by inhibiting the fungal cytochrome P450 (CYP51). KTZ is also a potent inhibitor of human cytochrome P450 3A4 (CYP3A4) enzyme, the major drug-metabolizing CYP isozyme in the human liver. We examined the enantioselective differences of KTZ in the inhibition of human CYP3A4 and in antifungal action. Dextro- and levo-KTZ exhibited modest enantioselective differences with respect to CYP3A4 inhibition of testosterone and methadone metabolism. For both substrates levo-KTZ was approximately a 2-fold more potent inhibitor. We examined the enantioselective differences in the in vitro activity of KTZ against medically relevant species of Candida and Aspergillus, as well as Cryptococcus neoformans. Overall, levo-KTZ was 2-4-fold more active than dextro-KTZ. Therefore, levo-KTZ is a more potent inhibitor of CYP3A4 and has stronger in vitro antifungal activity. Chirality 16:79-85, 2004.     

Metabolic activation of the pneumotoxin, 3-methylindole, by vaccinia-expressed cytochrome P450s

Twelve human cytochrome P450s and one mouse P450 were produced in HepG2 cells using vaccinia virus cDNA expression and analyzed for their ability to bioactivate the pneumotoxin, 3-methylindole (3MI), to an electrophilic metabolite(s) which alkylated cellular macromolecules. Cell lysates containing CYP2C8, CYP3A4, CYP2A6 and CYP2F1 metabolized 3MI to an intermediate(s) that became covalently bound to lysate material. A control lysate produced from cells which had been infected with a wild-type vaccinia virus was not able to bioactivate 3MI. The mouse 1A2 enzyme metabolized 3MI at a rate of 75.4 pmol/mg protein/minute, while the rate of metabolism in the lysate containing the human 1A2 P450 enzyme was not different from that in the control lysate. Therefore, the catalytic capabilities of orthologous P450 enzymes to activate 3MI cannot be extrapolated among different species. These results indicate that human P450s are capable of bioactivating 3MI to a metabolite which binds to cellular macromolecules suggesting that this compound may be toxic to humans.     

Structure-function analysis of human cytochrome P450 3A4 using 7-alkoxycoumarins as active-site probes

The oxidation of a series of seven alkyl ethers of 7-hydroxycoumarin by cytochrome P450 3A4 (CYP3A4) has been studied to probe the active site of the enzyme. TLC of the reaction mixture showed formation of metabolites other than 7-hydroxycoumarin. The separation and characterization of the different metabolites of the C4 to C7 compounds were achieved using a combination of TLC, HPLC, and gas chromatography-electron impact mass spectra. Among the 7-alkoxycoumarins, 7-hexoxycoumarin was found to be the most suitable candidate for investigating the active site of cytochrome CYP3A4, due to the well-separated metabolite peaks on TLC and HPLC. 7-hexoxycoumarin was found to produce three side-chain hydroxylated products besides 7-hydroxycoumarin: 7-(5-hydroxyhexoxy)coumarin, 7-(4-hydroxyhexoxy)coumarin, and 7-(3-hydroxycoumarin). The substitution of residues from substrate recognition sites -1, -4, -5, and -6 of CYP3A4 showed a strong influence on the product profile of 7-hexoxycoumarin, the most prominent effects observed with mutants at residues 119, 301, 305, 370, 373, and 479. The docking of 7-hexoxycoumarin into a molecular model of CYP3A4 also confirmed the presence of these residues within 5 A of the substrate. A comparative study of cytochrome P450 2B1 showed that the active-site mutants F206L, T302V, V363A, and S478G but not V363L exhibited a dramatic decrease in total 7-hexoxycoumarin hydroxylation. The study suggests that although the electronic nature of the substrate is important, enzymatic constraints significantly contribute to CYP3A4 selectivity.     

Design, synthesis, and antifungal activities of novel 1H-triazole derivatives based on the structure of the active site of fungal lanosterol 14 alpha-demethylase (CYP51)

A series of fluconazole (1) analogues, compounds 3a-k, were prepared as potential antifungal agents. They were designed by computational docking experiments to the active site of the cytochrome P450 14alpha-sterol demethylase (CYP51), whose crystal structure is known. Preliminary biological tests showed that most of the target compounds exhibit significant activities against the eight most-common pathogenic fungi. Thereby, the most potent congener, 1-[(4-tert-butylbenzyl)(cyclopropyl)amino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (3j), was found to exhibit a broad antifungal spectrum, being more active against Candida albicans, Candida tropicalis, Cryptococcus neoformans, Microsporum canis, and Trichophyton rubrum (MIC80 < 0.125 microg/ml) than the standard clinical drug itraconazole (2). The observed affinities of the lead molecules towards CYP51 indicate that a cyclopropyl residue enhances binding to the target enzyme. Our results may provide some guidance for the development of novel triazole-based antifungal lead structures.