Revisiting the mechanism of P450 enzymes with the radical clocks norcarane and spiro[2,5]octane

Norcarane (1) and spiro[2.5]octane (2) yield different product distributions depending on whether they are oxidized via concerted, radical, or cationic mechanisms. For this reason, these two probes were used to investigate the mechanisms of hydrocarbon hydroxylation by two mammalian and two bacterial cytochrome P450 enzymes. Products indicative of a radical intermediate with a lifetime ranging from 16 to 52 ps were detected during the oxidation of norcarane by P450(cam) (CYP101), P450(BM3) (CYP102), CYP2B1, and CYP2E1. Trace amounts of the cation rearrangement product were observed with norcarane for all but CYP2E1, while no cation or radical rearrangement products were observed for spiro[2.5]octane. The results for the oxidation of norcarane with a radical rearrangement rate of 2 x 10(8) s(-1) are consistent with the involvement of a two-state radical rebound mechanism, while for the slower (5 x 10(7) s(-1)) spiro[2,5]oct-4-yl radical rearrangement products were beyond detection. Taken together with earlier data for the hydroxylation of bicyclo[2.1.0]pentane, which also suggested a 50 ps radical lifetime, these three structurally similar and functionally simple substrates show a consistent pattern of rearrangement that supports a radical rebound mechanism for this set of cytochrome P450 enzymes.     

液相色谱-质谱联用技术结合多探针底物法研究人参皂苷Rb1的体外代谢

以奥美拉唑、 苯妥英、 卡马西平和非那西丁为检测肝药酶细胞色素P450酶(CYP450)亚型的专属探针药物, 通过原型药物减少量测定法考察药物体外代谢的变化, 评价人参皂苷Rb1对CYP450不同亚型酶的作用. 结果表明, P2C9, P2C19和P3A4实验组与对照组差异不显著, P1A2实验组与对照组差异显著, 表明人参皂苷Rb1能诱导P1A2亚型酶的活性, 促进底物与酶反应, 加快底物的代谢, 而对P2C9, P2C19和P3A4三个亚型酶有弱的诱导或无诱导作用. 根据快速分离液相色谱-质谱联用(RRLC-MS/MS)检测结果推断, 人参皂苷Rb1在CYP450酶中的代谢产物可转化为人参皂苷Rb1氧化产物(Rb1+O)及人参皂苷Rd和F2.     

The carbon-skeleton rearrangement in tropane alkaloid biosynthesis

High-level quantum chemistry calculations have been performed to examine the carbon-skeleton rearrangement of the tropane alkaloid littorine to hyoscyamine. Two pathways involving radical and carbocation intermediates have been investigated in this regard, namely, stepwise (or fragmentation-recombination) and concerted. The fragmentation products are calculated to be of high energy for both the radical- and carbocation-based mechanisms (136.3 and 170.9 kJ mol(-1), respectively). Similarly, the rearrangement barrier for the radical-based concerted pathway is calculated to be quite high (135.6 kJ mol(-1)). In contrast, the carbocation-based concerted pathway is found to be associated with a relatively low barrier (47.4 kJ mol(-1)). The ionization energy of the substrate-derived radical 3a is calculated to be 7.01 eV, suggesting that its oxidation to generate the substrate-derived carbocation 3b ought to be facile. In an attempt to investigate how an enzyme might modulate the rearrangement barriers, the separate and combined influences of partially protonating the migrating group and partially deprotonating the spectator OH group of the substrate were investigated. Such interactions can lead to significant reductions in the rearrangement barrier for both the radical- and carbocation-based concerted pathways, although the carbocation pathway continues to have significantly lower energy requirements. Also, the relatively high (gas-phase) acidity of the OH group of the product-related carbocation 4b indicates that the direct formation of hyoscyamine aldehyde (6) is a highly exothermic process. Although we would not wish to rule out alternative possibilities, our calculations suggest that a concerted rearrangement mechanism involving carbocations constitutes a viable low-energy pathway for the carbon-skeleton rearrangement in tropane alkaloid biosynthesis.     

Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119

CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.     

QM/MM modeling of benzene hydroxylation in human cytochrome P450 2C9

The mechanism of benzene hydroxylation was investigated in the realistic enzyme environment of the human CYP 2C9 by using quantum mechanical/molecular mechanical (QM/MM) calculations of the whole reaction profile using the B3LYP method to describe the QM region. The calculated QM/MM barriers for addition of the active species Compound I to benzene are consistent with experimental rate constants for benzene metabolism in CYP 2E1. In contrast to gas-phase model calculations, our results suggest that competing side-on and face-on geometries of arene addition may both occur in the case of aromatic ring oxidation in cytochrome P450s. QM/MM profiles for three different rearrangement pathways of the initially formed sigma-adduct, leading to formation of epoxide, ketone, and an N-protonated porphyrin species, were calculated. Our results suggest that epoxide and ketone products form with comparable ease in the face-on pathway, whereas epoxide formation is preferred in the side-on pathway. Additionally, rearrangement to the N-protonated porphyrin species was found to be competitive with side-on epoxide formation. This suggests that overall, the competition between formation of epoxide and phenol final products in P450 oxidation of aromatic substrates is quite finely balanced.     

The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug Flow Bioreactors for the Production of Drug Metabolites

Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly(methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: (1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and (2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process.     

Liquid chromatography/tandem mass spectrometric determination of inhibition of human cytochrome P450 isozymes by resveratrol and resveratrol-3-sulfate

trans-Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to both have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities, and to be a weak inhibitor of cytochrome P450 (CYP)3A4, which might have significance for drug-drug interactions. Since trans-resveratrol is rapidly converted in vivo to primarily trans-resveratrol-3-sulfate, a rapid, selective, and sensitive method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed to investigate human cytochrome P450 inhibition by trans-resveratrol-3-sulfate. Effects of trans-resveratrol and trans-resveratrol-3-sulfate on the metabolism of selective cytochrome P450 substrates (CYP1A2/ethoxyresorufin, CYP2C9/diclofenac, CYP2C19/(S)-mephenytoin, CYP2D6/bufuralol, CYP3A4/testosterone) were monitored using cDNA-expressed human recombinant isozymes. For method validation, LC/MS/MS was used to measure the inhibition of various cytochrome P450 isozymes by different concentrations (0-50 microM) of known selective inhibitors. IC(50) values of 3.2, 1.4, 8.9, 0.2, and 0.3 microM were obtained for the standard isozyme inhibitors CYP1A2/furafylline, CYP2C9/sulfaphenazole, CYP2C19/tranylcypromine, CYP2D6/quinidine, and CYP3A4/ketoconazole, respectively, which were in good agreement with literature values. trans-Resveratrol showed IC(50) values of 11.6 microM for CYP2C19 and 1.1 microM for CYP3A4, but the IC(50) values exceeded 50 microM for all the other CYP isozymes, which indicated no inhibition. No enzyme inhibition was observed for trans-resveratrol-3-sulfate. Our results indicate that trans-resveratrol is a marginal inhibitor of CYP3A4 and a weak inhibitor of CYP2C19, but its major metabolite trans-resveratrol-3-sulfate is not an inhibitor of any of the cytochrome P450 isozymes investigated.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine

Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.     

Genome mining in streptomyces. Discovery of an unprecedented P450-catalyzed oxidative rearrangement that is the final step in the biosynthesis of pentalenolactone

The penM and pntM genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and Streptomyces arenae TU?469 were predicted to encode orthologous cytochrome P450s, CYP161C3 and CYP161C2, responsible for the final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1). Synthetic genes optimized for expression in Escherichia coli were used to obtain recombinant PenM and PntM, each carrying an N-terminal His(6)-tag. Both proteins showed typical reduced-CO UV maxima at 450 nm, and each bound the predicted substrate, pentalenolactone F (4), with K(D) values of 153 ± 14 and 126 ± 11 μM for PenM and PntM, respectively, as determined by UV shift titrations. PenM and PntM both catalyzed the oxidative rearrangement of 4 to 1 when incubated in the presence of NADPH, spinach ferredoxin, ferredoxin reductase, and O(2). The steady-state kinetic parameters were k(cat) = 10.5 ± 1.7 min(-1) and K(m) = 340 ± 100 μM 4 for PenM and k(cat) = 8.8 ± 0.9 min(-1) and K(m) = 430 ± 100 μM 4 for PntM. The in vivo function of both gene products was confirmed by the finding that the corresponding deletion mutants S. exfoliatus/ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer produced pentalenolactone but accumulated the precursor pentalenolactone F. Complementation of each deletion mutant with either penM or pntM restored production of antibiotic 1. Pentalenolactone was also produced by an engineered strain of Streptomyces avermitilis that had been complemented with pntE, pntD, and either pntM or penM, as well as the S. avermitilis electron-transport genes for ferredoxin and ferrodoxin reductase, fdxD and fprD.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

The CYPome of Sorangium cellulosum So ce56 and identification of CYP109D1 as a new fatty acid hydroxylase

The first systematic study of the complete cytochrome P450 complement (CYPome) of Sorangium cellulosum So ce56, which is a producer of important secondary metabolites and has the largest bacterial?genome sequenced to date, is presented. We describe the bioinformatic analysis of the So ce56 cytochrome P450 complement consisting of 21 putative P450 genes. Because fatty acids play a pivotal role during the complex life cycle of myxobacteria, we focused our studies on the characterization of fatty acid hydroxylases. Three novel potential fatty acid hydroxylases (CYP109D1, CYP264A1, and CYP266A1) were used for detailed characterization. One of them, CYP109D1 was able to perform subterminal hydroxylation of saturated fatty acids with the support of two autologous and one heterologous electron transfer system(s). The kinetic parameters for the product hydroxylation were derived.     

Protein film electrochemistry of microsomes genetically enriched in human cytochrome p450 monooxygenases

This communication demonstrates direct electron delivery from electrodes to cyt P450 reductases in stable films ( approximately 100 nm thick) of genetically enriched CYP1A2 and CYP3A4 microsomes made by layer-by-layer assembly with polyions. Reversible voltammetry of films containing genetically enriched cyt P450 monooxygenase microsomes was shown to involve cyt P450 reductase by comparison with the pure rabbit reductase and by lack of characteristic reactions of iron heme enzymes, such as reaction of the FeII form with CO and catalytic electrochemical reduction of oxygen and hydrogen peroxide. The microsome films were activated electrochemically to catalyze styrene epoxidation, consistent with the pathway utilized in the human liver, although further work is required to establish this definitively.     

Modulating the coupling efficiency of human cytochrome P450 CYP3A4 at electrode surfaces through protein engineering

This study shows that regulating the electron flow to the heme of human cytochrome P450 CYP3A4, using artificial redox chains, can significantly enhance its coupling efficiency and catalytic activity at electrode surfaces. The human CYP3A4 was fused at the genetic level either to the reductase domain of CYP102A1 (BMR) to create the CYP3A4/BMR or to Desulfovibrio vulgaris flavodoxin (FLD) to create the CYP3A4/FLD. Direct electrochemistry of the CYP3A4, CYP3A4/BMR and CYP3A4/FLD on glassy carbon and gold electrodes showed that the BMR and FLD flavo-proteins reduced the electron transfer rate to the CYP3A4 heme. Electrocatalysis resulted in appreciably higher product formation with the immobilized CYP3A4/BMR and CYP3A4/FLD on both surfaces due to an increased coupling efficiency. Rotating disk electrode studies and quantification of hydrogen peroxide were consistent with the proposed mechanism of a longer lived iron-peroxy species in the immobilized CYP3A4/BMR and CYP3A4/FLD. The approaches in this study provide a better understanding of cytochrome P450 uncoupling at electrode surfaces and aids in the construction of improved cytochrome P450 biosensors and bioelectrocatalysts.     

P450 versus P420: correlation between cyclic voltammetry and visible absorption spectroscopy of the immobilized heme domain of cytochrome P450 BM3

Cyclic voltabsorptometry is used for the first time to distinguish and characterize electrochemically the active (P450) and inactive (P420) forms of cytochromes P450 immobilized on an electrode during voltammetry experiments. This was achieved by using the heme domain (BMP) of the bacterial cytochrome P450 BM3 from Bacillus megaterium (CYP102A1) immobilized on mesopouros tin-oxide (SnO2) electrodes. We demonstrate that the formation of either the P450 form or the P420 one can be obtained by modifying the mesoporous electrode surface with polycations with different properties such as polyethylenimmine (PEI) and polydiallyldimethylammonium chloride (PDDA). Potential step spectroelectrochemistry allowed measurement of reduction potentials of the active P450 form. Values of -0.39+/-0.01 V and -0.58+/-0.01 V (both versus Ag/AgCl) were calculated for the active P450 form immobilized on the BMP/PDDA-SnO2 and BMP/PEI-SnO2 electrodes, respectively. The cyclic voltabsorptometric experiments showed how, when both the active and inactive forms are present on the PEI film, the inactive P420 species tends to dominate the cyclic voltammetric signal.     

Direct electrochemistry of cytochrome P450 in a biocompatible film composed of an epoxy polymer and acetylene black

We report on a composite matrix composed of epoxy copolymers P (GMA-co-MPC) and acetylene black that can be used to entrap cytochrome P450. The composite provides a biocompatible microenviroment and can substantially accelerate the electron transfer between the cytochrome P450 and the electrode. The electrochemical response is characterized by a pair of well-defined redox peaks for the heme Fe(II/III) redox couples were observed at ?483?mV (vs. SCE). The immobilized cytochrome P450 exhibits excellent electrocatalytical activity to diethylstilbestrol (DES). The amperometric response varied linearly with the concentration of DES in the 0.2 to 2.8?μM concentration range. The biosensor displays a detection limit 5.9?×?10^-8?mol?L^-1 and thus represents a promising candidate for studying the electrochemistry of cytochrome P450s and its sensing applications.

Products of cytochrome P450(BioI) (CYP107H1)-catalyzed oxidation of fatty acids

[reaction: see text] Oxidation of tetradecanoic and hexadecanoic acids by cytochrome P450(BioI) (CYP107H1) produces mainly the 11-, 12-, and 13-hydroxy C(14) fatty acids and the 11- to 15-hydroxy C(16) fatty acids, respectively. In contrast to previous reports, terminal hydroxylation is not observed. The enantiospecificity of fatty acid hydroxylation by P450(BioI) was also determined, and the enzyme was shown to be moderately selective for production of the (R)-alcohols.     

Homology modeling, binding site identification and docking in flavone hydroxylase CYP105P2 in Streptomyces peucetius ATCC 27952

Homology models of cytochrome P450 105P2 (CYP105P2) were constructed using four P450 structures, CYP105A1, CYP105, CYP165B3 and CYP107L1, as templates for the model building. Using Accelrys Discovery Studio 2.1 software, the lowest energy CYP105P2 model was then assessed for stereochemical quality and side-chain environment. Further active site optimization of the CYP105P2 model built using these templates was performed by molecular dynamics to generate the final CYP105P2 model. The substrates, flavone, flavanone, quercetin and naringenin, were docked into the model. The model-flavone complex was used to validate the active site architecture, and structurally and functionally important residues were identified by subsequent characterization of the secondary structure.     

Inhibition of acyl-coenzyme A:cholesterol acyltransferase stimulates cholesterol efflux from macrophages and stimulates farnesoid X receptor in hepatocytes

We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).     

Stereoselective determination of propafenone enantiomers in transgenic Chinese hamster CHL cells expressing human cytochrome P450

An enantioselective assay for S(+)- and R(-)-propafenone in transgenic Chinese hamster CHL cells expressing human cytochrome P450 was developed. The method involved extraction of propafenone from the S9s incubates, and formation of propafenone diastereomeric derivatives with the chiral reagent 2,3,4, 6-tetra-O-beta-D-glucopranosyl isothiocyanate. Separation and quantitation of diastereomeric propafenone derivatives were carried out in a reverse-phase-HPLC system with UV detection. The assay was linear from 2 to 200 microg/mL for each enantiomer. The analytical method gave average recoveries of 97.5% and 97.0% for S(+)- and R(-)-propafenone, respectively. The limits of detection and quantitation for the method are 0.1 and 2.0 microg/mL for both S(+)- and R(-)-propafenone, respectively. The reproducibility of the assay was good (RSD <10%). The method allowed study of the depletion of S(+)- and R(-)-propafenone in transgenic Chinese hamster CHL cells expressing human cytochrome P450. The stereoselectivity of propafenone phase I metabolism via cDNA-expressed CYP3A4 was observed.