Costas Ioannides, BSc (University of Liverpool), PhD (University of Surrey), DSc (University of Liverpool) is currently Professor of Mechanistic Toxicology at the University of Surrey and is an established author in this area.

Cytochromes P450

Role in the Metabolism and Toxicity of Drugs and other Xenobiotics

By Costas Ioannides

The Royal Society of Chemistry

Copyright © 2008 Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-274-6

Contents

Part A,
Chapter 1 Cytochrome P450 Structure and Function: An Evolutionary Perspective David F.V. Lewis and Yuko Ito,
Chapter 2 Generation of Reactive Intermediates by Cytochromes P450 Hermann M. Bolt and Peter H. Roos,
Part B,
Chapter 3 The CYP1A Subfamily Bhagavatula Moorthy,
Chapter 4 The CYP1B Subfamily Morag C.E. McFadyen and Graeme I. Murray,
Chapter 5 The CYP2A Subfamily Hannu Raunio, Jukka Hakkola and Olavi Pelkonen,
Chapter 6 The CYP2B Subfamily Laurent Corcos and François Berthou,
Chapter 7 The CYP2C Subfamily Stephen S. Ferguson, Karen Black and Jonathan P. Jackson,
Chapter 8 The CYP2D Subfamily Ulrich M. Zanger,
Chapter 9 The CYP2E Subfamily Lowell C. Overton, Alice Hudder and Raymond F. Novak,
Chapter 10 The CYP2F, CYP2G and CYP2J Subfamilies Qing-Yu Zhang and Xinxin Ding,
Chapter 11 The CYP3 Family David J. Greenblatt, Ping He, Lisa L. von Moltke and Michael H. Court,
Chapter 12 The CYP4 Family Allan E. Rettie and Edward J. Kelly,
Part C,
Chapter 13 Receptor-Mediated Regulation of Cytochromes P450 Kouichi Yoshinari, Eric Tien, Masahiko Negishi and Paavo Honkakoski,
Chapter 14 Modulation of Cytochromes P450 by Phytochemicals Michael Murray,
Chapter 15 Cytochromes P450 in Cancer Therapeutics Thomas K.H. Chang,
Subject Index, 510,

CHAPTER 1

Cytochrome P450 Structure and Function: An Evolutionary Perspective

DAVID F.V. LEWIS AND YUKO ITO

Table of Contents

1.1 Introduction 4
1.2 Evolutionary Aspects 7
1.3 Binding Functions of P450 11
1.4 Substrate Binding and Selectivity 15
1.5 P450 Catalysis 24
1.6 Structural Modelling of P450s 29
1.6.1 Molecular Modelling and Dynamics of P450s 30
1.6.2 Lipophilicity Relationships in P450 Substrate Binding and Selectivity 35
1.7 Conclusions 37
Abbreviations 38
Acknowledgements 39
References 39

1.1 Introduction

The cytochromes P450 constitute a superfamily of haem-thiolate enzymes which are ubiquitous in nature. Figure 1.1 shows the many fields in which P450s play important roles, thus highlighting their relevance to several branches of biological science. During the course of evolution, the P450 structure developed to bind the following entities: oxygen, carbon-based substrates, a haem group and redox partners, such as an iron-sulphur redoxin, an NADPH-dependent FAD- and FMN-containing flavoprotein reductase and cytochrome b5. In eukaryotic P450 systems, a membrane phospholipid bilayer such as that present in the smooth endoplasmic reticulum is also able to bind, as summarised in Tables 1.1 and 1.2. Mitochondrial P450s, such as CYP11 in the adrenal cortex, retain some of the prokaryotic P450 characteristics in possessing an iron-sulphur redoxin (specifically adrenodoxin) as a redox partner rather than utilising an NADPH-dependent flavoprotein reductase. This finding suggests that the mitochondria and, indeed, other cell organelles may have had bacterial origins, as has been reported previously from RNA comparisons. Over 5000 P450 sequences have been reported (Osamu Gotoh, personal communication) and it appears that these enzymes are present in all five biological kingdoms. Consequently, P450s will have undergone modifications during the course of evolution in order to adapt to the changes in environmental and cellular conditions.

P450 structures are known from X-ray crystallographic determinations and a list of these is provided in Table 1.3; surprisingly, there are relatively few differences across species ranging from bacteria to mammalia. Such differences include orientations of the B’ and F helices, together with the extent of poly-peptide in the loop regions between certain helical motifs, such as those of the F-G loop and the H-I loop. Also, there is an additional N-terminal peptide of some 40 residues which serves as a membrane anchor in eukaryotic P450s, and this is absent in prokaryotic sequences. Furthermore, it is not only in the primary sequence but also in the spatial orientation of secondary structural elements with respect to the haem moiety that P450s can vary. This may explain why homology models do not always correctly reproduce the active site topographies encountered in the actual crystal structures, although a relatively high homology (ie. over 40%) should provide a fairly good match between homology models and crystal structures. Indeed, for enzymes of the CYP2C family, it is found that models of CYP2C8 and CYP2C9, based on the CYP2C5 template, do indeed match closely with the actual crystal structures, and the relationship between degree of fit and sequence homology is explored further in a following section.

The tertiary structure of P450 possesses certain well-conserved ion-pairs (such as the ExxR motif) and other features (like the polyproline motif near the N-terminus) which determine its overall folding pattern, together with various hydrogen bonded, π-π stacking and hydrophobic contacts between amino acid residues, some of which are associated with haem and substrate binding. However, certain conserved basic residues on the surface of the enzyme tend to be utilised for the binding of redox partners. These charged surface residues are largely conserved across the superfamily for redox partner binding, and this can be illustrated by the example of putidaredoxin (an iron-sulphur redoxin) binding to P450cam, and also that of adrenodoxin binding to its reductase where it is apparent that the redoxin fits closely within a depression on the reductase surface. The structure of P450 resembles a triangular prism in overall shape with the haem moiety located approximately at the centre of one triangular face. The haem group lies in a depression, which is ideally suited, in terms of shape and complementary surface residues, for the binding of redox partners such as redoxins, cytochrome b5 and reductase. However, there are certain subtle distinctions between the entire binding regions of these various redox partners and the corresponding P450 enzymes which may explain why certain redox partners tend to bind at particular surface locations on the P450 enzymes.

1.2 Evolutionary Aspects

It is generally thought that life emerged around 3.5 billion years ago, which is about a billion years after the Earth’s formation (~4.55 billion years ago) although it is possible that the development of life may have occurred even earlier. P450s are ubiquitous within living systems and the enzymes have been found in all biological kingdoms, but some Archaea (early pro-karyotes) such as Escherichia coli, do not appear to contain any P450 enzymes whatsoever. However, two P450s have been isolated and crystallised from the thermophilic bacterial species Sulfolobus solfataricus and Thermus thermophilus. These organisms are apparently able to exist in the hot sulphurous conditions encountered in the thermal vents of undersea volcanic fissures, which are thought to represent the likely environment for early life formation around 3.5 billion years ago. Interestingly, the presence of iron and sulphur in such oceanic vents may have assisted in the generation of proteins requiring such elements including, for example, cytochrome P450 and its iron-sulphur ferredoxin redox partner. Figure 1.2 represents an abbreviated phylogenetic tree for certain P450s, and shows how this accords with the general development of terrestrial biota.

As the oxygen levels in the atmosphere began to increase around 2.5 billion years ago, protective systems would have developed to ensure species survival in a more aerobic environment, and eurakyotes are thought to have emerged about 2.1 billion years ago. This would have afforded some protection from the deleterious effects of free oxygen, and it is also possible that an early role of P450 at this time may have been in the detoxification of O2 itself. In microbial species, P450s are involved in the biosynthesis of antibiotics, certain toxins and for the generation of secondary metabolites. The full characterisation of such activities is currently being investigated, particularly with respect to the development of novel therapeutic agents via protein engineering of bacterial P450 enzymes, such as those from various Streptomyces species.

With the development of metazoa, the endogenous roles of P450 changed to steroid biosynthesis, together with that of fatty acid and prostanoid/ eicosanoid metabolism. When animal species started to colonise land areas in the Devonian period about 400 million years ago, plants developed toxins to deter animal predators, and many of these toxic compounds are known to be synthesised in part via P450-mediated pathways. However, it is thought that animal species began to develop new P450 enzymes specifically for the detoxification of these harmful plant products, and a phylogenetic analysis of P450s across the superfamily appears to support this viewpoint. Insect species also developed P450s with detoxifying roles. For example, the black swallowtail butterfly, Papilio polyxenes, possesses a specific P450 (CYP6B1) for metabolising the plant toxin methoxsalen (xanthotoxin), which itself may have been biosynthesised via the mediation of P450 enzymes. Furthermore, another insect-plant co-evolutionary role of P450 emerged when flowering plants appeared about 125 million years ago, as various P450s are known to be responsible for the biosynthesis of flower pigments such as the anthocyanins. It is thought, therefore, that the exogenous roles of P450s may have developed over a geological timescale via a co-evolutionary process, commonly termed plant-animal ‘warfare’ where animals developed certain xenobiotic-metabolising P450s to detoxify the deleterious effects of plant toxins which had been biosynthesised to deter animal predators.

The huge diversity of P450 functionality may, therefore, have arisen from the increase in atmospheric oxygen which was harnessed for oxidative metabolism and biosynthesis. For P450 utilises the inherent chemical power of the dioxygen molecule and controls its activation via two consecutive reductions to peroxide, which probably represents the precursor for the active oxygen species that is inserted into carbon-based substrates. The development of P450’s changing roles mirrors the evolution of terrestrial biota as indicated in Figure 1.2, and it should also be recognised that periodic mass extinctions have played their part in the rise of the mammalia and, eventually, to mankind itself. For example, the elaboration of the CYP2 family occurred after the major global extinction event at the end of the Permian period, approximately 250 million years ago.

As mentioned previously (vide supra), P450s are not found in E. coli and other primitive anerobic bacteria (ie. the Archebacteria) although they are present in certain thermophiles. These may provide a possible clue to the origins of P450 in abyssal thermal vents where there would have been the relatively high concentrations of iron and sulphur required for the formation for haem-thiolate proteins, as well as the iron-sulphur redoxins which constitute the earliest form of P450 redox partner. The relatively high percentage and clustering of aromatic amino acid residues present in these thermophilic bacterial P450s (from Sulfolobus solfataricus and Thermus thermophilus) are thought to provide a likely explanation for the thermal and high-pressure stability of these enzymes, thus enabling them to tolerate such extreme environments as may have occurred extensively in early terrestrial prehistory. The substrates for these P450s remain to be determined but one can realistically assume that oxygen levels would have been relatively low during this stage of biological development, possibly at a fraction of 1%. When atmospheric oxygen levels eventually started to increase, perhaps P450 enzymes had some role in detoxifying oxygen before the development of other biological defense systems for dealing with reactive oxygen species (ROS). In evolutionary terms, it would appear that dioxygen was not in the abundance it is today for most of the development of biological systems. It is interesting to note that the only fungal P450 to have had its crystal structure determined is, in fact, a nitric oxide reductase (CYP55) from Fusarium oxysporum, which utilises only nitric oxide in a coupling reaction, without the use of oxygen, to form nitrous oxide. However, the vast majority of P450 reactions (of which there are over 50 different types) involve splitting of the dioxygen molecule and subsequent monooxygenation of substrates while the unusual P450 reactions include: reduction, desaturation, oxidative ester cleavage, ring expansion, ring formation, aldehyde scission, dehydration, isomerisation, ipso attack of aromatic rings, one-electron oxidation, coupling reactions, rearrangement of fatty acid and prostaglandin hydroperoxides, and oxidative deamination. The more common P450-catalysed reactions are: aliphatic and aromatic hydro-xylation, N-hydroxylation, N- and S-oxidation, O-, S- and N-dealkylation, aromatisation, dehalogenation, dehydro-halogenation, epoxidation, deformy-lation and the reduction of nitro compounds, N-oxides, quinones, epoxides, azo compounds and certain halogen compounds. It is in the mammalian P450s where a wide diversity of reactions are characterised and, to some extent, this is due to the relatively large number of P450s in such species, together with their various endogenous and exogenous roles.

In the human P450 complement, there is the well-documented situation known as genetic polymorphism, where genetic defects in individual P450s (mainly CYP2D6 and CYP2C19) can give rise to significant changes in metabolic capacity towards certain substrates, including a number of drugs in clinical use. Consequently, there is considerable current interest in the screening of novel compounds that are destined for human exposure, such that adverse drug reactions can be avoided.

1.3 Binding Functions of P450

The apoprotein in P450 performs a number of specific functions by virtue of its primary, secondary and tertiary structure; some of these will be described in the remaining sections of this chapter, and Table 1.2 provides a summary. It is evident that the unusual functionalities of P450 enzymes evolved during the course of biological development from prokaryotic to eukaryotic organisms. Being haemoproteins, P450s contain a haem prosthetic grouping which, in common with most haemoproteins, is able to bind dioxygen although, in haemthiolate enzymes like P450, oxygen becomes activated via the unique properties of the thiolate ligand. In addition to the iron-sulphur covalent linkage, the haem moiety is bound by two conserved basic amino acid residues which form ion-pairs with the two haem propionate sidechains. A generally well-conserved tryptophan residue in the C helix, normally encountered in most mammalian P450s, is able to form a hydrogen bond with one of the haem propionate head-groups. Furthermore, the relatively planar haem group is bound in an essentially hydrophobic cleft within the P450 protein, formed by an intersection of the I and L helices, where a number of complementary residues enter into hydrophobic contacts with the haem structure. Also, a generally well-conserved serine residue in the C helix, located fairly close to the haem group, is thought to represent a phosphorylation site for initiating haem degradation (reviewed in reference 1).

The Fe2+/Fe3+ redox potential in P450 is modulated by substrate binding such that reduction of the enzyme by its redox partner is facilitated. In an ideal system like that of P450cam (CYP101), the binding of camphor makes the Fe2+/Fe3+ redox potential become less negative, such that P450 lies on a potential gradient between those of NADH, putidaredoxin and dioxygen, as shown in Figure 1.3. This mechanism prevents reduction of P450cam in the absence of the camphor substrate and there is a degree of similarity to that encountered in other bacterial systems, together with most mammalian P450s, although the situation is not so clear-cut in the microsomal system. The binding of redox partners shows both similarities and differences between various P450s, depending on the type of system concerned. In P450cam(CYP101), a group of basic residues surrounding the proximal haem face appear to form electrostatic interactions with a complementary cluster of acidic sidechains in the iron-sulphur protein, putidaredoxin, which enables electron transfer from the Fe2S2 centre to the haem via the mediation of a C-terminal tryptophan residue in putidaredoxin. A well-conserved proximal phenyl-alanine in P450 is thought to facilitate electron transport to the haem group, possibly by aromatic π-π stacking interactions, and Phe350 in P450 is an example of this type of grouping. This residue is present in both prokaryotic and eukaryotic P450s, whereas it is possible that a conserved tryptophan (vide supra) which is present in many, but not all, microsomal P450s could have undergone transference from the ferredoxin redox partner during the course of evolution. However, there is also evidence to suggest that this tryptophan plays a role in haem binding rather than solely acting as a conduit for electrons during the reduction process.

(Continues…)Excerpted from Cytochromes P450 by Costas Ioannides. Copyright © 2008 Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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