Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.     

Intensity enhancement of intraligand singlet-triplet π-π transition for the coordinated β-diketonate in Ni(II) complexes with a nitroxide radical

Room temperature and low temperature magnetic circular dichroism (MCD) in the intraligand spin-forbidden singlet-triplet π-π^∗ transition for the coordinated β-diketonate ligands were observed for the β-diketonato Ni(II) complexes with a chelated imino or nitronyl nitroxide radical, but not for the β-diketonato Ni(II) complexes without the radical ligands. This is elucidated by the borrowing mechanism from the singlet-singlet π-π^∗ transition through the hypothetical interligand β-diketonate-to-radical charge transfer (LLCT) in contrast to the case of Cr(III) complexes.     

Oxoiron(IV) complexes of the tris(2-pyridylmethyl)amine ligand family: effect of pyridine α-substituents

The oxoiron(IV) complexes of two 6-substituted tris(2-pyridylmethyl)amine ligand derivatives have been generated and characterized with respect to their spectroscopic and reactivity properties. The introduction of an α-substituent maintains the low-spin nature of the oxoiron(IV) unit but weakens the ligand field, as evidenced by red shifts in its characteristic near-IR chromophore. While its hydrogen-atom abstraction ability is only slightly affected, the oxo-transfer reactivity of the oxoiron(IV) center is significantly enhanced relative to that of the parent complex. These results demonstrate that the ligand environment plays a key role in modulating the reactivity of this important biological oxidant. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. An erratum to this article can be found at     

Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).     

X-ray photochemistry in iron complexes from Fe(0) to Fe(IV) - Can a bug become a feature?

Under intense soft X-ray irradiation, we have observed time-dependent changes in the soft X-ray spectra of virtually all the Fe coordination complexes that we have examined, indicating chemical transformation of the compound under study. Each compound, with oxidation states ranging from Fe(IV) to Fe(0), has been studied with either Fe L-edge spectroscopy or N K-edge spectroscopy. We find that very often a well-defined spectroscopic change occurs, at least initially, which is apparently capable of straightforward interpretation in terms of X-ray induced photoreduction, photooxidation or ligand photolysis. We briefly discuss the probable chemical nature of the changes and then estimate the rate of chemical change, thereby establishing the necessary radiation dose. We also demonstrate that the photochemistry not only depends on the Fe oxidation state but also the coordination chemistry of the complex. It seems that a proper understanding of such X-ray photochemical effects could well greatly assist the assignment of soft X-ray spectra of uncharacterized metal sites.     

Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens

Azoles have been applied widely to combat pathogenic fungi in medicine and agriculture and, consequently, loss of efficacy has occurred in populations of some species. Often, but not always, resistance was found to result from amino acid substitutions in the molecular target of azoles, 14α-sterol demethylase (CYP51 syn. ERG11). This review summarizes CYP51 function, evolution, and structure. Furthermore, we compare the occurrence and contribution of CYP51 substitutions to azole resistance in clinical and field isolates of important fungal pathogens. Although no crystal structure is available yet for any fungal CYP51, homology modeling using structures from other origins as template allowed deducing models for fungal orthologs. These models served to map amino acid changes known from clinical and field isolates. We conclude with describing the potential consequences of these changes on the topology of the protein to explain CYP51-based azole resistance. Knowledge gained from molecular modeling and resistance research will help to develop novel azole structures.     

Co (II), Ni(II) and Cu(II) complexes with a new pendant armed macrocyclic ligand showing several π,π-interactions

A new ligand, L, bearing four cyanoethyl pendant groups has been synthesized by reaction of the precursor ligand L¹ with acrylonitrile. The X-ray crystal structure of ligand L reveals the presence of a nanotubular structure in the solid state connected by intermolecular π,π-stacking interactions between adjacent pyridine rings. The coordination capability towards transition metal ions [Co(II), Ni(II) and Cu(II)] has been investigated starting from the hydrated nitrate and perchlorate salts of the metals. The new ligand L and the metal complexes obtained were characterized by elemental analysis, FAB MS, conductivity measurements, magnetic studies, IR and UV-vis spectroscopy. Furthermore, the crystal structure of ligand L and of the complexes [CoL][Co(NO₃)₄] · CH₃CN (1), [NiL](NO₃)₂ (3), [NiL](ClO₄)₂ · CH₃CN · 3H₂O (4), [CuL][Cu(NO₃)₃(H₂O)₂](NO₃) · H₂O (5) and [CuL](ClO₄)₂ · 2CH₃CN (6) were determined. The nitrate ions in the complexes are located near the pyridine rings and π,π-stacking interactions between pyridine rings, nitrate ions and nitrile groups have been found.     

Anticooperativity in diffusion-controlled reactions with pairs of anisotropic domains: a model for the antigen–antibody encounter

The encounter between anisotropic agents in diffusion-controlled reactions is a topic of very general relevance in chemistry and biology. Here we introduce a simplified model of encounter of an isotropic molecule with a pair of partially reacting agents and apply it to the encounter reaction between an antibody and its antigen. We reduce the problem to the solution of dual series relations, which can be solved iteratively, yielding the exact solution for the encounter rate constant at any desired order of accuracy. We quantify the encounter effectiveness by means of a simple indicator and show that the two binding centers systematically behave in an anti-cooperative fashion. However, we demonstrate that a reduction of the binding active sites allows the composite molecule to recover binding effectiveness, in spite of the overall reduction of the rate constant. In addition, we provide a simple formula that enables one to calculate the anti-cooperativity as a function of the size of the binding site for any values of the separation between the two active lobes and of the antigen size. Finally, some biological implications of our results are discussed.     

Isolation and phylogeny of novel cytochrome P450 genes from tunicates (Ciona spp.): a CYP3 line in early deuterostomes?

Cytochromes P450 (CYPs) form a gene superfamily involved in the biotransformation of numerous endogenous and exogenous natural and synthetic compounds. In humans, CYP3A4 is regarded as one of the most important CYPs due to its abundance in liver and its capacity to metabolize more than 50% of all clinically used drugs. It has been suggested that all CYP3s arose from a common ancestral gene lineage that diverged between 800 and 1100 million years ago, before the deuterostome-protostome split. While CYP3s are well known in mammals and have been described in lower vertebrates, they have not been reported in non-vertebrate deuterostomes. Members of the genus Ciona belong to the tunicates, whose lineage is thought to be the most basal among the chordates, and from which the vertebrate line diverged. Here we describe the cloning, exon-intron structure, phylogeny, and estimated expression of four novel genes from Ciona intestinalis. We also describe the gene structure and phylogeny of homologous genes in Ciona savignyi. Comparing these genes with other members of the CYP clan 3, show that the Ciona sequences bear remarkable similarity to vertebrate CYP3A genes, and may be an early deuterostome CYP3 line.     

Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan – Identification of mutants that dehalogenate 3-chloroindole

Cytochrome P450_cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450_cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate. Mutant (E156G/V247F/V253G/F256S) had the highest maximal velocity in the conversion of 3-chloroindole to isatin, whereas mutants (T56A/N116H/D297N) and (G60S/Y75H) had highest k_cat/K_M values. Six of the mutants had more than one mutation, and within this set, mutation of residues 297 and 179 was observed twice. Docking simulations were performed on models of the mutant enzymes; the wild-type did not accommodate 3-chloroindole in the active site, whereas all the mutants did. We propose two potential reaction pathways for dechlorination of 3-chloroindole. 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.     

Is there a toxicological advantage for non-hyperbolic kinetics in cytochrome P450 catalysis? Functional allostery from "distributive catalysis"

The cytochrome P450s (CYPs) are the major enzymatic detoxification and drug metabolism system. Recently, it has become clear that several CYP isoforms exhibit positive and negative homotropic cooperativity. However, the toxicological implications of allosteric kinetics have not been considered, nor understood. The allosteric kinetics are particularly enigmatic in several respects. In many cases, CYPs bioactivate substrates to more toxic products, thus making it difficult to rationalize a functional advantage for positive cooperativity. Also, CYPs exhibit cooperativity with many structurally diverse ligands, in marked contrast to the specificity observed with other allosteric systems. Here, kinetic simulations are used to compare the probabilistic time- and concentration-dependent integrated toxicity function during conversion of substrate to product for CYP models exhibiting Michaelis-Menten (non-cooperative) kinetics, positive cooperativity, or negative cooperativity. The results demonstrate that, at low substrate concentrations, the slower substrate turnover afforded by cooperative CYPs compared with Michaelis-Menten enzymes can be a significant toxicological advantage, when toxic thresholds exist. When present, the advantage results from enhanced "distribution" of toxin in two pools, substrate and product, for an extended period, thus minimizing the chance that either exceeds its toxic threshold. At intermediate concentrations, the allosteric kinetics can be a modest advantage or modest disadvantage, depending on the kinetic parameters. However, at high substrate concentrations associated with a high probability of toxicity, fast turnover is desirable, and this advantage is provided also by the cooperative enzymes. For the positive homotropic cooperativity, the allosteric kinetics minimize the probability of toxicity over the widest range of system parameters. Furthermore, this apparent functional cooperativity is achieved without specific molecular recognition that is the hallmark of "traditional" allostery.     

ω-Hydroxylation of α-tocopheryl quinone reveals a dual function for cytochrome P450-4F2 in vitamin E metabolism

α-Tocopherol (α-TOH) is the primary lipophilic radical trapping antioxidant in human tissues. Oxidative catabolism of α-tocopherol (αTOH) is initiated by ω-hydroxylation of the terminal carbon (C-13) of the isoprenoid sidechain followed by oxidative transformations that sequentially truncate the chain to yield the 2,5,7,8-tetramethyl(3′carboxyethyl)-6-hydroxychroman (α-CEHC). After conjugation to glucuronic acid, 3′-carboxyethyl-6-hydroxychroman glucuronide is excreted in urine. We report here that the same enzyme that accomplishes this task, the cytochrome P450 monooxygenase CYP-4F2, can also ω-hydroxylate the terminal carbon of α-tocopheryl quinone. A standard sample of ω-OH-α-tocopheryl quinone (ω-OH-α-TQ) was synthesized as a mixture of stereoisomers by allylic oxidation of α-tocotrienol using SeO₂ followed by double-bond reduction and oxidation to the quinone. After incubating human liver microsomes or insect cell microsomes expressing only recombinant human CYP-4F2, cytochrome b5, and NADPH P450 reductase with d₆-α-tocopheryl quinone (d₆-αTQ), we showed that the ω-hydroxylated (13-OH) d₆-α-TQ was produced. We further identified the production of the terminal carboxylic acid d₆-13-COOH-αTQ. The ramifications of this discovery to the understanding of tocopherol utilization and metabolism, including the quantitative importance of the αTQ-ω-hydroxylase pathway in humans, are discussed.     

Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.     

Oxidation-state-dependent reactions of cytochrome <Emphasis Type="Italic">c</Emphasis> with the trioxidocarbonate(•1−) radical: a pulse radiolysis study

The reaction of the trioxidocarbonate(*1-) radical (CO (3) (*-) , "carbonate radical anion") with cytochrome c was studied by pulse radiolysis at alkaline pH and room temperature. With iron(III) cytochrome c, CO (3) (*-) reacts with the protein moiety with rate constants of (5.1 +/- 0.6) x 10(7) M(-1) s(-1) (pH 8.4, I approximately 0.27 M) and (1.0 +/- 0.2) x 10(8) M(-1) s(-1) (pH 10, I = 0.5 M). The absorption spectrum of the haem moiety was not changed, thus, amino acid radicals produced on the protein do not reduce the haem. The pH-dependent difference in rate constants may be attributed to differences in ionization states of amino acids and to the change in the conformation of the protein. With iron(II) cytochrome c, CO (3) (*-) oxidizes the haem quantitatively, presumably via electrostatic guidance of the radical to the solvent-accessible haem edge, with a different pH dependence: at pH 8.4, the rate constant is (1.1 +/- 0.1) x 10(9) M(-1) s(-1) and, at pH 10, (7.6 +/- 0.6) x 10(8) M(-1) s(-1). We propose that CO (3) (*-) oxidizes the iron center directly, and that the lower rate observed at pH 10 is due to the different charge distribution of iron(II) cytochrome c.     

Hepatic morphological alterations,glycogen content and cytochrome P450 activities in rats treated chronically with N<Superscript>ω</Superscript>-nitro-L-arginine methyl ester (L-NAME)

Chronic treatment of rats with N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) biosynthesis, results in hypertension mediated partly by enhanced angiotensin-I-converting enzyme (ACE) activity. We examined the influence of L-NAME on rat liver morphology, on hepatic glycogen, cholesterol, and triglyceride content, and on the activities of the cytochrome P450 isoforms CYP1A1/2, CYP2B1/2, CYP2C11, and CYP2E1. Male Wistar rats were treated with L-NAME (20 mg/rat per day via drinking water) for 2, 4, and 8 weeks, and their livers were then removed for analysis. Enzymatic induction was produced by treating rats with phenobarbital (to induce CYP2B1/2), beta-naphthoflavone (to induce CYP1A1/2), or pyrazole (to induce CYP2E1). L-NAME significantly elevated blood pressure; this was reversed by concomitant treatment with enalapril (ACE inhibitor) or losartan (angiotensin II AT(1) receptor antagonist). L-NAME caused vascular hypertrophy in hepatic arteries, with perivascular and interstitial fibrosis involving collagen deposition. Hepatic glycogen content also significantly increased. L-NAME did not affect fasting glucose levels but significantly reduced insulin levels and increased the insulin sensitivity of rats, based on an intraperitoneal glucose tolerance test. Immunoblotting experiments indicated enhanced phosphorylation of protein kinase B and of glycogen synthase kinase 3. All these changes were reversed by concomitant treatment with enalapril or losartan. L-NAME had no effect on hepatic cholesterol or triglyceride content or on the basal or drug-induced activities and protein expression of the cytochrome P450 isoforms. Thus, the chronic inhibition of NO biosynthesis produced hepatic morphological alterations and changes in glycogen metabolism mediated by the renin-angiotensin system. The increase in hepatic glycogen content probably resulted from enhanced glycogen synthase activity following the inhibition of glycogen synthase kinase 3 by phosphorylation.     

Inhibitor and substrate activities of sesquiterpene olefins toward (+)-δ-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1)

Several lines of evidence indicate that (+)-δ-cadinene-8-hydroxylase (CYP706B1) plays an important role in biosynthesis of gossypol in Gossypium arboreum L. ( [Luo et al., 2001] and [Wang et al., 2003]). The catalytically active enzyme has been expressed in yeast microsomes. Some microsomal preparations conjugated the hydroxylated (+)-δ-cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-β-d-glucoside (OG), a non-ionic detergent, (+)-δ-cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-δ-cadinene. OG had little effect on K_m and slightly stimulated apparent V_max. Enzymic activity was more than 10-fold more sensitive to inhibition by the N-substituted imidazole clotrimazole than to miconazole. Sesquiterpene olefins (−)-δ-cadinene, (−)-α-cubebene, (−)-α-muurolene, α-humulene, and a mixture of (−)- and (+)-α-copaene were inhibitory to hydroxylation of (+)-δ-cadinene. In addition, (−)-α-cubebene, (−)-α-muurolene, α-humulene, and, to a smaller extent, (−)-δ-cadinene served as alternative substrates for (+)-δ-cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, α-humulene and α-copaene are found in lysigenous glands of cotton (Elzen et al., 1985), which are also the site of gossypol accumulation ( [Bell et al., 1978] and [Mace et al., 1976]) and the probable site of its biosynthesis.     

De novo sequence analysis of cytochrome P450 1–3 genes expressed in ostrich liver with highest expression of CYP2G19

The cytochrome P450 (CYP) 1–3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1–3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2–10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.