Target-assembled tandem oligonucleotide systems based on exciplexes for detecting DNA mismatches and single nucleotide polymorphisms

We report the first exciplex-based split-probe system for DNA detection. The detector is split at a molecular level into signal-silent components which, before a signal is generated, must be assembled correctly into a particular three-dimensional arrangement. The model system comprises of two 8-mer oligonucleotides, complementary to neighbouring sites of a 16-mer DNA target, each equipped with moieties able to form an exciplex on correct, contiguous hybridization. The exciplex emits at approximately 480 nm with a large Stokes shift (135 nm). The extremely rigorous structural demands for exciplex formation and emission were achieved by careful structural design and by the discovery that high levels of certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplex, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. Inserts and mismatches can be effectively detected by this exciplex construct giving potential for single nucleotide polymorphism detection.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems.

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

Cholesterol amperometric biosensor based on cytochrome P450scc

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min).     

Characterization of cytochrome P450 2D6 alleles using the Invader system

The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.     

Induction of cytochrome P450 by toluene

At least six cytochrome P450 (P450) isoenzymes, including CYP1A1/2, CYP2A1, CYP2B1/2, CYP2C6, CYP2C11 and CYP2E1, are involved in the metabolism of toluene in rat liver. Toluene exposure induces CYP1A1/2, CYP2B1/2, CYP2E1 and CYP3A1, but decreases CYP2C11/6 and CYP2A1 in adult males. Both sex and age influence the induction of P450s by toluene: in general, the inductive effect is more prominent in younger than in older animals; in males than in females. Neonatal exposure to toluene causes significant changes in liver microsomal P450 dependent monooxygenase activities during the early stage of life, whereas the enects on the rats of more than 3 weeks of age are small. Although structurally related chemicals of toluene also influence similar hepatic P450 isoenzymes, the degree of CYP2B1/2 induction increases, whilst that of CYP2E1 decreases with increasing molecular weight and aliphatic moieties. Unlike liver, exposure to toluene does not influence the distribution of pulmonary or renal microsomal P450-related enzyme activity in rats. In humans, occupational exposure to toluene is so low that it could not lead to the induction of P450. However, the induction may be seen in toluene sniffers who are exposed to high concentrations.     

Oxygen activation by cytochrome P450 monooxygenase

Unlike photosystem II (PSII) that catalyzes formation of the O-O bond, the cytochromes P450 (P450), members of a superfamily of hemoproteins, catalyze the scission of the O-O bond of dioxygen molecules and insert a single oxygen atom into unactivated hydrocarbons through a hydrogen abstraction-oxygen rebound mechanism. Hydroxylation of the unactivated hydrocarbons at physiological temperatures is vital for many cellar processes such as the biosynthesis of many endogenous compounds and the detoxification of xenobiotics in humans and plants. Even though it carries out the opposite of the water splitting reaction, P450 may share similarities to PSII in proton delivery networks, oxygen and water access channels, and consecutive electron transfer processes. In this article, we review recent advances in understanding the molecular mechanisms by which P450 activates dioxygen.     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

Oxidation of human cytochrome P450 1A2 substrates by Bacillus megaterium cytochrome P450 BM3

Cytochrome P450 enzymes (P450s or CYPs) are good candidates for biocatalysis in the production of fine chemicals, including pharmaceuticals. Despite the potential use of mammalian P450s in various fields of biotechnology, these enzymes are not suitable as biocatalysts due to their low stability, low catalytic activity, and limited availability. Recently, wild-type and mutant forms of bacterial P450 BM3 (CYP102A1) from Bacillus megaterium have been found to metabolize various. It has therefore been suggested that CYP102A1 may be used to generate the metabolites of drugs and drug candidates. In this report, we show that the oxidation reactions of typical human CYP1A2 substrates (phenacetin, ethoxyresorufin, and methoxyresorufin) are catalyzed by both wild-type and mutant forms of CYP102A1. In the case of phenacetin, CYP102A1 enzymes show only O-deethylation product, even though two major products are produced as a result of O-deethylation and 3-hydroxylation reactions by human CYP1A2. Formation of the metabolites was confirmed by HPLC analysis and LC–MS to compare the metabolites with the actual biological metabolites produced by human CYP1A2. The results demonstrate that CYP102A1 mutants can be used for cost-effective and scalable production of human CYP1A2 drug metabolites. Our computational findings suggest that a conformational change in the cavity size of the active sites of the mutants is dependent on activity change. The modeling results further suggest that the activity change results from the movement of several specific residues in the active sites of the mutants.     

Oxidation of indole by cytochrome P450 enzymes

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Rearrangement reactions catalyzed by cytochrome P450s

Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants.     

Ethylbenzene hydroxylation by cytochrome P450cam.

The metabolism of ethylbenzene by cytochrome P450cam was analyzed by experimental and theoretical methods. The present experiments indicate that ethylbenzene is hydroxylated almost exclusively at the secondary ethyl carbon with about a 2:1 ratio of R:S product. Several molecular dynamics trajectories were performed with different starting conformations of ethylbenzene in the active site of P450cam. The stereochemistry of hydroxylation predicted from the molecular dynamics simulations was found to be in good agreement with the observed products.     

Regulation of cytochrome P450 expression by sphingolipids

Sphingolipids modulate many aspects of cell function, including the expression of cytochrome P450, a superfamily of heme proteins that participate in the oxidation of a wide range of compounds of both endogenous (steroid hormones and other lipids) and exogenous (e.g. alcohol, drugs and environmental pollutants) origin. Cytochrome P450-2C11 (CYP 2C11) is down-regulated in response to interleukin-1beta (IL-1beta), and this response involves the hydrolysis of sphingomyelin to ceramide as well as ceramide to sphingosine, and phosphorylation of sphingosine to sphingosine 1-phosphate. Activation of ceramidase(s) are a key determinant of which bioactive sphingolipid metabolites are formed in response to IL-1beta. Ceramidase activation also appears to account for the loss of expression of CYP 2C11 when hepatocytes are placed in cell culture, and the restoration of expression when they are plated on Matrigel; hence, this pathway is influenced by, and may mediate, interactions between hepatocytes and the extracellular matrix. Recent studies using inhibitors of sphingolipid metabolism have discovered that sphingolipids are also required for the induction of CYP1A1 by 3-methylcholanthrene, however, in this case, the requirement is for de novo sphingolipid biosynthesis rather than the turnover of complex sphingolipids. These findings illustrate how changes in sphingolipid metabolism can influence the regulation of at least several isoforms of cytochrome P450.     

Examining the mechanism of stimulation of cytochrome P450 by cytochrome b5: the effect of cytochrome b5 on the interaction between cytochrome P450 2B4 and P450 reductase

Dissociation constants K(d) for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b(5) (b(5)) stimulates metabolism of this substrate and dramatically decreases the substrate-related K(d) values. Experiments are performed to test if the b(5)-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b(5) (Mn b(5)) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b(5) stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b(5) is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b(5), with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.     

New perspectives on the conformational equilibrium regulating multi-phasic reduction of cytochrome P450 2B4 by cytochrome P450 reductase

The pre-steady-state reduction of cytochrome P450 (P450) 2B4 by P450 reductase (reductase) was modeled by assuming that an equilibrium between three catalytic conformers of P450 regulates the multi-phasic reduction of the enzyme. This model was compared to a model of reduction involving a minimum number of phases. Based on several criteria, the former model seems to provide an improved fit to the reduction data. Substrates were divided into two groups based on their effects at different concentrations of reductase. Surprisingly, in the presence of some substrates (group 1) but not others (group 2), the rate of reduction was actually slower with an excess of reductase than with equimolar reductase and P450. Presumably, oxidized reductase binds differently to P450 than reduced reductase. A schematic model based on two sites of interaction between reductase and P450 2B4 is offered to explain the unusual reduction kinetics with the two different groups of substrates.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

Keeping the spotlight on cytochrome P450

This review describes the recent advances utilizing photosensitizers and visible light to harness the synthetic potential of P450 enzymes. The structures of the photosensitizers investigated to date are first presented along with their photophysical and redox properties. Functional photosensitizers range from organic and inorganic complexes to nanomaterials as well as the biological photosystem I complex. The focus is then on the three distinct approaches that have emerged for the activation of P450 enzymes. The first approach utilizes the in situ generation of reactive oxygen species entering the P450 mechanism via the peroxide shunt pathway. The other two approaches are sustained by electron injections into catalytically competent heme domains either facilitated by redox partners or through direct heme domain reduction. Achievements as well as pitfalls of each approach are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Phosphorylation of cytochrome P450: regulation by cytochrome b5

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.