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Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3‐step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C−C bond scission (removing the 19‐oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2−, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen‐18 labeling, i.e. from 18O2 and H218O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19‐oxo‐androstenedione by human P450 19A1 and of a model secosteroid, 3‐oxodecaline‐4‐ene‐10‐carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non‐enzymatic acid‐catalyzed deformylation, yielding 19‐norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e. Fe3+O2−) in both P450 19A1 oxidation of 19‐oxo‐androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016–16025), attributed to several technical modifications. [ABSTRACT FROM AUTHOR]

The 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2−) or Compound I (perferryl oxygen, FeO3+) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18O incorporation into formic acid from 18O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18O (one atom) into formic acid, consistent with a major but not exclusive FeO2− mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X‐ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Å away from the heme iron atom. Experiments with human P450 51A1 and H218O yielded primarily one 18O atom but 14 % of the formic acid product with two 18O atoms, indicative of a minor contribution of a Compound I mechanism. LC–MS evidence for a Compound 0‐derived Baeyer–Villiger reaction product (a 14α‐formyl ester) was also found. [ABSTRACT FROM AUTHOR]

To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-20-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol j Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 30-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat, suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair. [ABSTRACT FROM AUTHOR]

A combination of chemical modifications and LC‐tandem MS was used for the structure elucidation of various ethylene crosslinks of DNA with O6‐alkylguanine‐DNA alkyltransferase (AGT, see picture). The elucidation of the chemical structures of such DNA–protein crosslinks is necessary to understand mechanisms of mutagenesis. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes account for ~75% of the metabolism of drugs. Most of the reactions catalyzed by P450s are mixed-function oxidations, and a C-H bond is (usually) broken. The rate-limiting nature of this step can be analyzed using the kinetic isotope effect (KIE) approach. The most relevant type of KIE is one termed intermolecular non-competitive, indicative of rate-limiting C-H bond breaking. A plot of KIE versus kcat for several P450s showed a correlation coefficient ( r2) of 0.62. Deuterium substitution has been considered as a potential means of slowing drug metabolism or redirecting sites of metabolism in some cases, and several general points can be made regarding the potential for application of deuterium in drug design/development based on what is known about P450 KIEs. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Crystal structures of a binary Mg2+-form Dpo4-DNA complex with 1, N2-etheno-dG in the template strand as well as of ternary Mg2+-form Dpo4-DNA-dCTP/dGTP complexes with 8-oxoG in the template strand have been determined. Comparison of their conformations and active-site geometries with those of the corresponding Ca2+-form complexes revealed that the DNA and polymerase undergo subtle changes as a result of the catalytically more active Mg2+ occupying both the A and B sites. [ABSTRACT FROM AUTHOR]

Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23–33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1′-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes catalyze a variety of oxidation and some reduction reactions, collectively involving thousands of substrates. A general chemical mechanism can be used to rationalize most of the oxidations and involves a perfenyl intermediate (FeO3+) and odd-electron chemistry, i.e. abstraction of a hydrogen atom or electron followed by oxygen rebound and sometimes rearrangement. This general mechanism can explain carbon hydroxylation, heteroatom oxygenation and dealkylation, epoxidation, desaturation, heme destruction, and other reactions. Another approach to understanding catalysis involves analysis of the more general catalytic cycle, including substrate specificity, because complex patterns of cooperativity are observed with several P450s. Some of the complexity is due to slow conformational changes in the proteins that occur on the same timescale as other steps. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:163-168, 2007; Published online in Wiley InterScience (). DOI 10.1002/jbt.20174 [ABSTRACT FROM AUTHOR]

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-β-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes include a family of related enzymes that are involved in metabolism of vitamins, steroids, fatty acids, and other chemicals. This review presents a brief historical overview of the discovery and characterization of P450 enzymes extending from intermediary metabolism to the fields of drug metabolism and toxicology. Introductions to P450 enzyme structure and function are also presented. The goals of this review are to 1) provide an introduction to a few of the many aspects of P450 research relating to humans, 2) introduce additional ways of thinking about metabolism, 3) provide some basic examples of P450 enzymology, and 4) provide applications to topics widely taught in undergraduate courses in biochemistry. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. [Copyright &y& Elsevier]

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6β-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6β- and 2β-hydroxy products, was identified as 1β-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of≈ 6 µg of this product. Mass spectrometry established the formula as C19H29O3 (MH+ 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and theβ-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1α-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes. [ABSTRACT FROM AUTHOR]

The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries. [ABSTRACT FROM AUTHOR]

The basis for our previous observations [Kaminsky, L. S., Guengerich, F. P., Dannan, G. A. & Aust, S. D. (1983) Arch. Biochem. Biophys. 225, 398–404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4′, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver eytochrome P-450 (P-450) isozymes reconstituted with NADPH-cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P-450UT-F > P-450PB-D ≥ P-450UT-A ≥ P-450BNF/ISF-G > P-450PB/PCN-E > P-450PB-B ≥ P-450PB-C ≥ P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P-450 isozymes. The results suggest that the reductase and P-450 isozymes may be located differently relative to one another in the various microsomal preparations. [ABSTRACT FROM AUTHOR]

Hepatocytes from adult and newborn humans were put into primary culture and exposed to phenobarbital, 3-methylcholanthrene, or rifampicin, three well-known inducers of cytochrome P.450 in animals. The expression of four cytochrome P450 enzymes (or groups of enzymes, namely P450 IIIA. P-450 IICS/9/10, P450 lIE1, and P-450 1A2) was investigated. These enzymes were found to remain expressed during the period of culture studied. Treatment with the inducers for three days resulted in different responses. depending upon the inducer and the enzyme. Phenobarbital and rifampicin increased P-450 11C89/10 mRNA transcripts and the corresponding protein, while 3-methytcholanthrene was ineffective. Both P-450 IIlA mRNA and protein were strongly induced by rifampicin. All of the hepatocytes were found to synthesize P-450 IIlA in response to rifampicin, as shown by immunoperoxidase staining. P-450 IIlA expression was not affected by phenobarbital and was decreased by 3- methyicholanthrene. P-450s 1A2 and IIE1 decreased to 25- 50% of the initial level during these cultures. P-450 1A2 and ethoxyresorufin O-deethylase activity (which is a menooxygenase activity related to P.450 IA family) were increased only by 3-methyleholanthrene and did not respond to the other inducers. P-450 IIEI was not induced by any of these compounds. P-450 IIC8/9/10 and P450 lIlA mRNA levels were also measured in human hepatocytes from one newborn. P450 11C8/9/10 was barely expressed in freshly isolated cells but increased dramaticaily with time in culture. P.450 IIlA transcripts were abundant in both freshly isolated and cultured cells derived from a newborn. These results clearly demonstrate that human hepatocytes continue to express cytochrome P450 enzymes and respond to inducers in culture. This model system provides a useful approach for investigating the effects of drugs on maturation and expression of drug-metabolizing enzymes in human liver. [ABSTRACT FROM AUTHOR]

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity, Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16β-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16α increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus. [ABSTRACT FROM AUTHOR]

The composition of isozymes of hepatic microsomal cytochrome P-450 (P- 450) from C57BL/6 (B6) and DBA/2 (D2) inbred strains of mice, uninduced and treated with phenobarbital (PB), pregnenolone-16α-carbonitrile (PCN), and β-naphthoflavone (BNF), were assessed. The assessment was based on comparisons of the regioselectivity and stereoselectivity of warfarin metabolism, inhibition of metabolism by antibodies raised against highly purified rat P-450 isozymes (the specificity of these antibodies in inhibiting rat microsomal metabolism of warfarin was also determined), and by immunoelectrophoresis using the anti-(rat P-450). Isozymes were considered to be the same or very similar if they exhibited the same substrate specificity, antigenicity, and molecular weight. Untreated D2 and B6 mice had similar compositions of P-450 isozymes which were independent of the Ah receptor. Both strains contained an isozyme very similar to rat P- 450UT -A. PB or PCN induction of the two mouse strains proceeded independently of the Ah receptor and yielded isozymes which are the same as or very similar to the rat P- 450UT -A, P- 450PB -B, P- 450PB -C, and P- 450PB /PCN-E isozymes. D2 and B6 mice had similar isozyme compositions when treated with PB or PCN, but isozyme compositions varied in differently induced mice. BNF treatment of the D2 mice was without effect, but the B6 mice produced an isozyme(s) which was different from rat P- 450BNF -B, the major BNF-induced isozyme in rat liver. [ABSTRACT FROM AUTHOR]

Focuses on the synthesis and insertion, both in vivo and in vitro, of rat-liver cytochrome P-450 and epoxide hydratase into xenopus laevis membranes. Role played by liver microsomal enzymes cytochrome P-450 in the metabolism of xenobiotics and endogenous compounds; Details of a study on the topic. [ABSTRACT FROM AUTHOR]

© 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:173-174, 2005; Published online in Wiley InterScience (). DOI 10.1002/jbt.20072 [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to an article on the U.S. Environmental Protection Agency's reliance on a biologically-based model of rat liver tumor induction by dioxin.

Schering 66712 (SCH66712) is a known mechanism-based inhibitor of human cytochrome P450 2D6. Spectral binding assays with purified recombinant protein indicate that SCH66712 displays classical Type I binding with ferric P450 2D6 with KS of 0.39± 0.10 µM. Incubation of SCH66712 with purified 2D6 and NADPH resulted in a loss of P450 heme-CO difference spectrum. Pyridine hemochrome assays also showed loss of heme over time. SDS-PAGE analysis of inactivation assays with human liver microsomes, recombinant microsomes or reconstituted purified 2D6 demonstrated that 3H- and 14C-labeled SCH66712 were irreversibly bound to the apoprotein in SCH66712 inactivated samples and that the association was NADPH dependent. ESI-LC-MS analysis of the SCH66712 inactivated samples showed that the mass of the inactivated 2D6 apoprotein increased by approximately 380 Da (SCH66712, 338 Da). [ABSTRACT FROM AUTHOR]

Sulfolobus solfataricus DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-2′-oxodeoxyguanosine (8-oxoG) in a highly efficient and accurate manner (∼95% C, ∼5% A incorporation). Previously, crystal structures revealed a potential role for Arg332 in stabilizing the anti conformation of the 8-oxoG template base by way of a hydrogen bond with the O8 atom, which results in an increase in enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8oxoG and dATP less favorable. In the present study, site-directed mutagenesis was used to replace Arg332 with Ala, Glu, Leu, or His to elucidate the importance of the hydrogen bond between Arg332 and the 08 atom of the modified guanine during Dpo4-catalyzed bypass of 8-oxoG. All four mutants insert both C and A opposite 8-oxoG (LC-MS/MS); the range of A incorporation observed was ∼17-25%. Transient-state kinetic results suggest that Glu332 retains fidelity against bypass of 8-oxoG, most similar to wild type Dpo4. A crystal structure of the Glu332 mutant and 8-oxoG:A Hoogsteen pair reveals a water-mediated hydrogen bond between Glu332 and the N2 exocyclic amine group of adenine, suggesting an increased propensity for A insertion, consistent with the LC-MS and kinetic analyses. However, Glu332 is still the most accurate of the four mutants tested at bypass of 8-oxoG. These results lend support to the idea that an electrostatic interaction between Arg332 and 8-oxoG does indeed play a role in determining the fidelity of Dpo4-catalyzed bypass of the lesion. [ABSTRACT FROM AUTHOR]