BBA - General Subjects (v.1770, #3)
Editorial Board (ii).
Special issue: Cytochrome P450 by M.J. Coon (313).
Complex reactions catalyzed by cytochrome P450 enzymes by Emre M. Isin; F. Peter Guengerich (314-329).
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.
Keywords: Cytochrome P450; Oxidation reactions; Rearrangement reactions; Bioactivation; Metabolism of drugs;
Cytochrome P450 systems—biological variations of electron transport chains by Frank Hannemann; Andreas Bichet; Kerstin M. Ewen; Rita Bernhardt (330-344).
Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.
Keywords: Cytochrome P450; Electron transfer; Heme-thiolate protein; Monooxygenase; Redox partner; P450 system;
Cytochrome P450–redox partner fusion enzymes by Andrew W. Munro; Hazel M. Girvan; Kirsty J. McLean (345-359).
The cytochromes P450 (P450s) are a broad class of heme b-containing mono-oxygenase enzymes. The vast majority of P450s catalyse reductive scission of molecular oxygen using electrons usually derived from coenzymes (NADH and NADPH) and delivered from redox partner proteins. Evolutionary advantages may be gained by fusion of one or more redox partners to the P450 enzyme in terms of e.g. catalytic efficiency. This route was taken by the well characterized flavocytochrome P450BM3 system (CYP102A1) from Bacillus megaterium, in which soluble P450 and cytochrome P450 reductase enzymes are covalently linked to produce a highly efficient electron transport system for oxygenation of fatty acids and related molecules. However, genome analysis and ongoing enzyme characterization has revealed that there are a number of other novel classes of P450–redox partner fusion enzymes distributed widely in prokaryotes and eukaryotes. This review examines our current state of knowledge of the diversity of these fusion proteins and explores their structural composition and evolutionary origins.
Keywords: Cytochrome P450; Redox partner; Fusion protein; Flavoprotein; Ferredoxin; Electron transfer; Catalytic cycle; Phthalate dioxygenase reductase; Flavodoxin; Peroxidase; Chimera; Heme binding; Linker; Enzyme mechanism; Domain;
Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling) by Shakunthala Narasimhulu (360-375).
The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O2 and specific electron transport system. The ternary complex S–Fe+O2 with substrate and O2 bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L6 position of the iron (Site II) to which O2 binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L6 position of the iron. The substrate camphor binds to Site I, and releases the L6 water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its –OH group forming Fe–O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub-sites are also reflected in the data on binding of the substrate camphor (S) in the presence of the product 5-exo-OH camphor (P) to CYP101 (E). The data are in accordance with the two-site model involving the ternary complex ESP. The affinity of the substrate to the product-bound enzyme as well as the affinity of the product to the substrate-bound enzyme decreased with increase in product concentration, which is consistent with mixed inhibition indicative of inhibitory allosteric interactions between the two sub-sites. Implications of these observations for coupling/uncoupling mechanisms are discussed in the light of the published findings consistent with the two-site behavior of the P450 active site. In addition, kinetic data indicating that the transient high spin intermediate may have to be taken into account for understanding how some P450s have been able to express appreciable hydroxylation activities in the absence of substrate-induced low to high spin transition, observable by the traditional static spectroscopy, are presented.
Keywords: Substrate; Product; Cytochrome; P450; Sub-site; Coupling; Uncoupling;
What common structural features and variations of mammalian P450s are known to date? by Michal Otyepka; Josef Skopalík; Eva Anzenbacherová; Pavel Anzenbacher (376-389).
Sufficient structural information on mammalian cytochromes P450 has now been published (including seventeen X-ray structures of these enzymes by June 2006) to allow characteristic features of these enzymes to be identified, including: (i) the presence of a common fold, typical of all P450s, (ii) similarities in the positioning of the heme cofactor, (iii) the spatial arrangement of certain structural elements, and (iv) the access/egress paths for substrates and products, (v) probably common orientation in the membrane, (vi) characteristic properties of the active sites with networks of water molecules, (vii) mode of interaction with redox partners and (viii) a certain degree of flexibility of the structure and active site determining the ease with which the enzyme may bind the substrates. As well as facilitating the identification of common features, comparison of the available structures allows differences among the structures to be identified, including variations in: (i) preferred access/egress paths to/from the active site, (ii) the active site volume and (iii) flexible regions. The availability of crystal structures provides opportunities for molecular dynamic simulations, providing data that are apparently complementary to experimental findings but also allow the dynamic behavior of access/egress paths and other dynamic features of the enzymes to be explored.
Keywords: Cytochrome P450; CYP; CYP structure; Molecular dynamic; Active site; Substrate binding;
The ins and outs of cytochrome P450s by Vlad Cojocaru; Peter J. Winn; Rebecca C. Wade (390-401).
The active site of cytochromes P450 is situated deep inside the protein next to the heme cofactor. Consequently, enzyme specificity and kinetics can be influenced by how substrates pass through the protein to access the active site and how products egress from the active site. We previously analysed the channels between the active site and the protein surface in P450 crystal structures available in October 2003 [R.C. Wade, P.J. Winn, I. Schlichting, Sudarko, A survey of active site access channels in cytochromes P450, J. Inorg. Biochem. 98 (2004) 1175–1182]. Since then, 52 new P450 structures have been made available, including entries for ten isozymes for which structures were not previously available. We present an updated survey covering all P450 crystal structures available in March 2006. This survey shows channels not observed earlier in crystal structures, some of which were identified in previous molecular dynamics simulations. The crystal structures demonstrate how some of the channels can merge when the protein structure opens up resulting in a wide cleft to the active site, caused largely by movements of the F–G helix–loop–helix and the B–C loop. Significant differences were observed between the channels in the crystal structures of the mammalian and bacterial enzymes. The multiplicity of channels suggests possibilities for substrate channelling to and from the P450s.
Keywords: Cytochrome P450; Protein dynamics; Ligand binding; Substrate access channel; Water channel;
Structure–function analysis of cytochromes P450 2B by Yonghong Zhao; James R. Halpert (402-412).
In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure–function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.
Keywords: Cytochrome P450; CYP2B; Structure; Function; Substrate specificity; Conformation;
Structural analysis of cytochromes P450 shows differences in flexibility of heme 2- and 4-vinyls by Jiří Hudeček; Petr Hodek; Eva Anzenbacherová; Pavel Anzenbacher (413-419).
Structural analysis of the orientations of heme vinyl side chains was carried out using the published crystallographic data for different cytochromes P450. Torsional angles (τ, CαCβ–CaCb) show different distributions for the vinyls in positions 2 and 4. Whereas the orientation of 2-vinyls is rather restricted (τ between − 120° and − 180°), the 4-vinyls have a much higher mobility over almost the entire conformational space. On the basis of the empirical correlation recently reported for peroxidases (M.P. Marzocchi, G. Smulevich, Relationship between heme vinyl conformation and the protein matrix in peroxidases, J. Raman Spectrosc. 34 (2003), 725–736), an attempt has been made to compare the observed vinyl orientations with the experimental frequencies of the vinyl stretching vibrational modes. The data for P450 proteins do not exactly match the peroxidase-derived function, although a qualitatively similar relationship is likely to exist. Differences between P450 forms suggest a variability in heme-region flexibility and in communication with the rest of enzyme.
Keywords: Cytochromes P450; Heme vinyls; Resonance Raman spectra; Enzyme structural flexibility;
FTIR studies of the redox partner interaction in cytochrome P450: The Pdx–P450cam couple by Andrey Karyakin; Domantas Motiejunas; Rebecca C. Wade; Christiane Jung (420-431).
Recently we have developed a new approach to study protein–protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam–Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP–FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in β-sheets and α-helix content, a decrease in the population of random coil/310-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam–Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx–P450cam complex.
Keywords: Infrared spectroscopy; Protein–protein interaction; Salt bridge; Principal component analysis; Cytochrome P450cam; Putidaredoxin;
Nitric-oxide synthase: A cytochrome P450 family foster child by Antonius C.F. Gorren; Bernd Mayer (432-445).
Nitric-oxide synthase (NOS), the enzyme responsible for mammalian NO generation, is no cytochrome P450, but there are striking similarities between both enzymes. First and foremost, both are heme-thiolate proteins, employing the same prosthetic group to perform similar chemistry. Moreover, they share the same redox partner, a diflavoprotein reductase, which in the case of NOS is incorporated with the oxygenase in one polypeptide chain. There are, however, also conspicuous differences, such as the presence in NOS of the additional cofactor tetrahydrobiopterin, which is applied as an auxiliary electron donor to prevent decay of the oxyferrous complex to ferric heme and superoxide. In this review similarities and differences between NOS and cytochrome P450 are analyzed in an attempt to explain why NOS requires BH4 and why NO synthesis is not catalyzed by a member of the cytochrome P450 family.
Keywords: Nitric-oxide synthase; Tetrahydrobiopterin; Uncoupling;
A combinatorial approach to substrate discrimination in the P450 CYP1A subfamily by Valérie Taly; Philippe Urban; Gilles Truan; Denis Pompon (446-457).
A comparison of all known mammalian CYP1A sequences identifies nineteen sequence regions that are conserved within all 1A1s or within all 1A2s but at the same time systematically differ between any 1A1 and any 1A2. The purpose of this study was to explore links between these specific CYP1A sequence signatures and substrate specificity shift through the kinetic analysis of combinatorial variants of increasing complexity. The less complex variants correspond to multiple mutations within a short segment of their sequence. The more complex variants correspond to mosaic P450s recombining 1A1 and 1A2 sequences (up to 5 crossovers per sequence). Fifty-eight such functional CYP1A variants and parental wild-type enzymes were expressed in yeast and assayed with 7-alkoxyresorufins and ethoxyflurorescein ethyl ester as substrates. Observed kinetic data were analyzed by multivariate statistical analyses and hierarchical clustering in order to highlight correlations and identify potential sequence–activity relationships within the three-dimensional function space investigated. Several variants are outliers in these representations and show a redistribution of their substrate specificity compared to wild-type CYP1As. Some combinations of sequence elements were identified that significantly discriminate between 1A1 and 1A2 for these three substrates. The comparison of this combinatorial approach with previous results of site-directed mutagenesis is discussed.
Keywords: Multivariate statistic; CYP1A1; CYP1A2; Mutant; DNA shuffling; Random mutagenesis; Resorufin; Fluorescein; ProSAR;
Mammalian cytochromes P450—Importance of tissue specificity by Matej Seliskar; Damjana Rozman (458-466).
Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.
Keywords: Cytochrome P450; Endoplasmic reticulum; Signal sequence; Mammal; Subcellular localization; Tissue distribution;
Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms by Galina I. Lepesheva; Michael R. Waterman (467-477).
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.
Keywords: Sterol 14α-demethylase (CYP51); Sterol biosynthesis; Substrate preferences; Catalytic activity; Inhibition;
The human cytochrome P450 sub-family: Transcriptional regulation, inter-individual variation and interaction networks by Nick Plant (478-488).
The Cytochrome P450 super-family is a fundamental requirement for the viability of most life, with Cytochrome P450 proteins having been identified in organisms ranging from bacteria to man. These enzymes may be subdivided into those that metabolise purely endogenous chemicals, and those that are involved in xenobiotic metabolism. Of the latter group it can be argued that CYP3A sub-family members rank as the most important; their high expression in the liver and wide substrate specificity mean that they are clinically important in the metabolism of many therapeutic drugs, and alteration in their activity is central to many clinically-relevant drug–drug interactions. In this review I will examine the human CYP3A enzymes, discussing their genome structure, common allelic variants and, in greatest detail, their transcriptional regulation. Through examination of these characteristics we will see both striking similarities and differences between the four human CYP3A enzymes, which may have important impacts on inter-individual response to chemical exposure. Finally, the role of nuclear receptors in regulating CYP3A gene expression, and indeed that of many other proteins involved in drug metabolism, will be examined: Such an examination will show the need to utilize a systems biology approach to understand fully how the human body responds to chemical exposure.
Keywords: CYP3A; Gene expression; PXR; CAR; Nuclear receptor; Inter-individual variability; Drug–drug interactions;
Clinical implications of pharmacogenetics of cytochrome P450 drug metabolizing enzymes by Julia Kirchheiner; Angela Seeringer (489-494).
For many drugs, pharmacogenetic polymorphisms are known affecting biotransformation and clinical outcome. The clinical importance of these variants depends on allele-frequency and the effect size of the clinical outcome parameters. Further, it depends on the therapeutic range of the drug which is affected, on predictability of drug response as well as on duration until onset of therapeutic efficacy. Consequences which arise from genotyping might be: adjustment of dose according to genotype, choice of therapeutic strategy or even choice of drug. In antidepressant drug treatment, most drugs are metabolized via the polymorphic cytochrome P450 enzyme CYP2D6. Huge differences in pharmacokinetic parameters have been consistently shown for many tricyclics, some SSRIs, and other antidepressant drugs whereas the effects on therapeutic efficacy and adverse events have been described controversially. In cardiovascular disease, oral anticoagulants, nonsteroidal anti-inflammatory drugs, oral hypoglycemic drugs and other drugs are affected by genetic polymorphisms of the cytochrome P450 drug metabolizing enzyme CYP2C9. Studies in patients or healthy volunteers revealed up to 10-fold differences in pharmacokinetic parameters due to genetic polymorphisms of CYP2C9. Pharmacogenetics based dose adjustments are one tool to individualize drug treatment according to genetic factors. They can be derived from pharmacokinetic data with the aim to obtain equal drug concentrations in each individual. Prospective validation of dose adjustments based on pharmacogenetics should be performed before routine application of such strategies. A controlled prospective clinical trial with one arm receiving genotype-based dose adjustments and the other arm receiving therapy as usual will elucidate the benefit of pharmacogenomics-based individualization of certain drug therapies.
Keywords: Cytochrome P450; Genotype based drug dosing; CYP2D6; CYP2C9;