J COON,’ XINXIN Department

and

progress

N4rneP45O:

DING, STEVENJ.

of Biological Chemistrc

The University

The cytochrome P450 gene superfamily encodes many isoforms that are unusual in the variety of chemical reactions catalyzed and the number of substrates attacked. The latter include physiologically important substances such as steroids, eicosanoids, fatty acids, lipid hydroperoxides, retinoids, and other lipid metabolites, and xenobiotics such as drugs, alcohols, procarcinogens, antioxidants, organic solvents, anesthetics, dyes, pesticides, odorants, and flavorants. Accordingly, it is not surprising that these catalysts have come under intensive study in recent years in fields as diverse as biochemistry and molecular biology, endocrinology, pharmacology, toxicology, anesthesiology, nutrition, pathology, and oncology. In this review, recent advances in our knowledge of the catalytic properties, reaction mechanisms, and regulation of expression and activity of the P450 enzymes are briefly summarized. In addition, the prospects for research in this field are considered, and advances are predicted in four broad areas: improved basic knowledge of enzyme catalysis and regulation; synthesis of fine chemicals, including drug design and screening; removal of undesirable environmental chemicals; and biomedical applications related to steroid, drug, carcinogen, and alcohol metabolism. Coon, M. J.; Ding, X.; Pernecky, S. J.; Vaz, A. D. N. Cytochrome P450: progress and predictions. FASEBJ. 6: 669-673; 1992. ABSTRACT

-

Key Wo,th: of expression

ytochrome P450 Lrozjines oxygen activation . lipid peroxidation . olefin formation from

regulation aldehydes

P450 HAS BECOME THE subject of intensive research in recent years in many laboratories for two reasons. An understanding of the remarkable versatility of this family of enzymes is of interest to those studying biological catalysis from a fundamental point of view, and an elucidation of the reactions catalyzed has obvious biomedical relevance for intestigators in endocrinology pharmacology, toxicology, anesthesiology, nutrition, pathology, oncology, and related fields. In this brief review, we have summarized recent advances that have led to our present knowledge of the multiplicity of P450 isoforms, substrates, catalytic mechanisms, and regulatory pathways and have predicted some future developments. CYTOCHROME

DIVERSITY

OF REACTIONS

piedictions

CATALYZED

What is now often called the P450 gene superfamily encodes numerous enzymes, of which more than 150 have so far been characterized. These vary from about 10 to over 90% in sequence identity and occur in biological sources as diverse as microorganisms, plants, and animals. Almost all mammalian tissues contain one or more of these cytochromes in various organelles, predominantly in the endoplasmic reticulum and mitochondria. Some of the P450 isoforms are fairly specific in their choice of substrates (for example, the steroidogenic cytochromes), but many, and particularly

ALPIN of Michigan

Medical

School,

D. N. VAZ Ann Arbor,

those in the hepatic endoplasmic reticulum, catalyze a surprisingly large number of chemical reactions with an almost unlimited number of biologically occurring and xenobiotic compounds (1-6). In the latter category are synthetic environmental chemicals, now estimated at about 250,000, most of which are potential P450 substrates if not inducers or inhibitors of the individual cytochromes. Examples of xenobiotics that serve as P450 substrates are drugs (including antibiotics), procarcinogens, antioxidants, organic solvents, anesthetics, dyes, pesticides, alcohols, odorants, and flavorants, and a variety of unusual substances in plants and microorganisms, which, despite their biological occurrence, are foreign to animals. Many new drugs and other organic compounds that will be synthesized in the future can also be expected to be substrates. The physiologically important substrates include steroids, eicosanoids, fatty acids, lipid hydroperoxides, retinoids, acetone, and acetol.

SYSTEMATIC

NOMENCLATURE

When definitive evidence was first obtained by enzyme fractionation and characterization for multiple forms of P450 (7), it was not known whether different species and tissues would have similar isoforms. The trivial names assigned by various investigators were based on the sources used or on spectral properties, electrophoretic mobility, substrates, or inducers, or in some instances numbers or letters were assigned in series. Chloroperoxidase and P450s have some physicochemical and catalytic similarities (8) but have no antigenic determinants in common (9). Standard methods of enzyme nomenclature based on the reactions catalyzed proved to be inadequate because various laboratories were studying different substrates, and in some cases different products or even different chemical reactions with the same cytochrome. With rapid advances in knowledge about P450s, and in particular about their amino acid sequences determined directly or predicted from the corresponding cDNAs, it became clear that a general nomenclature based on divergent evolution (as judged by structural homology) would be helpful. Thanks to Dr. Daniel W. Nebert, who proposed such a system and enlisted others in the effort, a nomenclature with the following guidelines has been widely adopted (10). Those P450 proteins from all sources with 40% or greater sequence identity are included in the same family, as designated by an Arabic number, and those with greater than 55% identity are then included in the same subfamily, as designated by a capital letter. The individual genes (and gene products) are then arbitrarily assigned numbers. There are now 28 families, of which 11 are predicted to exist in all mammals. As an example, the major phenobarbital-

‘To whom correspondence should be addressed, Biological Chemistry, The University of Michigan Ann Arbor, MI 48109, USA. of

at: Department Medical School,

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inducible cytochrome in rabbit liver microsomes, originally called P4SOLM2 or form 2 (7), has been assigned to family 2 and subfamily B, and the gene and the enzyme are designated CYP2B4 and CYP2B4, respectively. The enzyme may also be called P450 2B4. The main advantage of the unified nomenclature is that structurally identical or highly similar P450s are easily recognizable regardless of the sources from which they were isolated including the species, tissue, and organelle, or the inducer administered to the animal, or any of a variety of catalytic activities examined. Those outside the P450 field may find that designations such as CYP2B4, CYP3A1O, and CYP1O5C1 are confusing, but the unusual complexity of this enzyme family requires a systematic nomenclature, and it is to be hoped that investigators will also state the sources, inducers, and catalytic activities so as to aid readers of their publications. The unsuitable alternative was a series of trivial names; for example, LM3a, j, MKj1, Hj, alcohol oxygenase, aniline hydroxylase, N-nitrosodimethylamine demethylase, etc., as were used for what is now called P450 2E1. The designation P450 is still used, even though it was coined to describe a red pigment of unknown function having a reduced CO-difference spectrum with a major band at about 450 nm (11) and later shown to be involved in the oxidation of drugs and steroids (12). Even the term cytochrome is unsuitable, as in most reactions these catalysts function as oxygenases rather than as electron carriers.

DIVERSITY

OF

CATALYTIC

MECHANISMS

The steps involved in the P450-catalyzed reduction of molecular oxygen with incorporation of one oxygen atom into a substrate, RH, to give the corresponding product,

ROH,

are shown in Fig. 1. The scheme

is based on one pro-

posed earlier (13) with several modifications. More recent reviews (5, 6, 14) have summarized newer findings on substrate and peroxide activation, and an insightful review by White (15) has emphasized the involvement of free radicals

+

LO

in the mechanism of action of P450 and other monooxygenases. Also shown in the scheme is the release of products of 02 reduction that are not coupled to substrate hydroxylation, such as superoxide, hydrogen peroxide, and in the 4-electron NADPH oxidase reaction, water. The well-known peroxide shunt, in which a peroxy compound such as an alkyl hydroperoxide or peracid donates the oxygen atom for substrate hydroxylation with no requirement for molecular oxygen or for NADPH as an electron donor, is also shown. Much remains to be learned about factors controlling regio- and stereospecificity in P450-catalyzed reactions; the availability of specifically altered proteins from site-directed mutagenesis will be particularly useful in this respect. Although spectral (16) and EPR analysis (17) have provided evidence for activated oxygen intermediates, chemical and physical approaches are needed to identify these elusive species in detail. As reviewed elsewhere (6), some interesting variations on the reactions shown in Fig. 1 are the proposal of a cage radical mechanism for the rearrangement of a prostaglandin endoperoxide to a prostacyclin and a thromboxane (18), of radical intermediates in dehydrogenation reactions (14), and of aminium radical intermediates in amine oxidations (19). Ortiz de Montellano and Reich (20) have reviewed the unusual properties that permit P450 to contribute to the regulation of its own activities; these include competitive inhibition by many alternative substrates, some cases of mechanism-based inactivation, and stimulation or inhibition by compounds that serve as effectors. Also shown in the scheme is the ability of ferrous P450 to donate electrons in a stepwise fashion to bring about reactions under anaerobic conditions. Although P450 is widely recognized to be an oxygenating catalyst, less emphasis has been placed on its role as a reducing catalyst. Many compounds, including dyes, N-oxides, and epoxides undergo stepwise 2-electron reduction. Another example we have been investigating is the reductive cleavage of xenobiotic hydroperoxides and lipid hydroperoxides (shown as R’ LOOH in Fig. 1) with hydrocarbon formation (21). For example, cumyl hydroperoxide yields acetophenone and methane, and the hydroperoxide derived from linoleic acid (l3-hydroperoxy-9,11-octadecadienoic acid) yields 13-oxo-9,1ltridecadienoic acid (LO in the scheme) and pentane. The cleavage reaction is believed to involve stepwise 1-electron transfer, resulting in homolysis of the peroxide oxygenoxygen bond and generation of an alkoxy radical, with fiscission of the latter followed by reduction of the secondary radical

The alcohol-inducible form of P450 (form 2E1) is the most active isozyme examined in this reaction (22). We have suggested that P450 2E1, in addition to its known damaging effects in chemical toxicity and chemical carcinogenesis, may enhance the reductive cleavage of lipid hydroperoxides with a resultant loss in membrane integrity. The likely importance of P450 2El-dependent lipid peroxidation in vivo after ethanol abuse has also been pointed out by Ekstr#{246}mand Ingelman-Sundberg (23). More recently, we have described the P450-dependent conversion of cyclohexane carboxylaldehyde to cyclohexene with loss of the aldehyde carbon as formate, as shown on the lower left in Fig. 1 (24). This reaction may be a useful model for the demethylation reactions catalyzed by the steroidogenic P450s, aromatase and lanosterol demethylase, in which an olefinic product and formate are also formed (25, 26). The reaction requires P450, NADPH-cytochrome P450 reductase, NADPH, and 02. Externally added H202 is active with P450 in the deformylation reaction in the absence liver

Figure 1. Overall scheme for mechanism of action of P450. Fe represents the heme iron atom in the active site, RH a substrate, and ROH the corresponding monooxygenation product. R’ LOOH represents a lipid hydroperoxide and RH and LO represent the corresponding reduction products (alkane and oxoacid, respectively). XOOH represents a peroxy compound that serves as an alternate oxygen donor to molecular oxygen.

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FASEB lournal

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cytochrome

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of NADPH and the reductase. In contrast, iodosobenzene is ineffective, indicating that iron oxene is not the oxidant, and m-chloroperbenzoic acid and cumyl hydroperoxide are also inactive, which indicates that deformylation by H202 is mechanistically distinct from hydroxylation reactions supported by these oxidants. We have concluded that a peroxyhemiacetal-like adduct may be formed between the substrate and molecular oxygen-derived hydrogen peroxide. A role for oxygen-derived peroxide in the P450-catalyzed demethylation of steroids has been proposed by several research groups (27-32). Many other aldehydes also undergo this cleavage reaction, particularly those with branched carbon chains such as citronellal, which is found in many essential oils and is widely used as an odorant and flavorant (33). Shown in Fig. 2 is a scheme in which an enzyme-based peroxyhemiacetal-like intermediate, presumably formed from the heme iron-bound peroxide and the electrophilic aldehyde carbonyl group, rearranges to yield the olefin and formic acid by either a concerted or a sequential 13-scission mechanism.

DIVERSITY

OF REGULATORY

MECHANISMS

Interest in the regulation of cytochrome P450 originally stemmed from observations that the administration of structurally diverse chemicals results in induction, that is, an increase in the level of one or more isoforms. Categorization of these

inducers

is based

on the

cytochrome

that

is increased

in amount (34). Thus, polycyclic aromatic hydrocarbons induce P450 IA, whereas phenobarbital, glucocorticoids, ethanol, and clofibrate increase the levels of P450s 2B, 3A, 2E, and 4A, respectively, but examples are also known where

a single agent induces

two or more cytochromes

and where

several compounds induce the same cytochrome. The inductive response of 1AI to TCDD is mediated by a cytosolic receptor that binds ‘TCDD and then interacts with regulatory regions in the 5 flanking region of the gene to activate transcription, thereby leading to increased accumulation of 1A1 mRNA and an increase in the level of 1A1 in the endoplasmic reticulum (35, 36). Modulation of the transcriptional activity of the gene is the most common, but not the only, regulatory mechanism of P450 expression. In fact, transcriptional and diverse posttranscriptional mechanisms have been described for the control of P450 2E1 expression. The 2E1 gene is transcription-

Concerted: FeOOf

OH

-

Ht!0H

-

0 HOH

+

ci::I:J +

Fe-OH

Stepwise: Fe_O)H

[ I

H]]

+

-

Fe-OH

2e.. H 4

+

Figure 2. Proposed mechanism tion

of cyclohexane

CYTOCHROME

P450: ADVANCES

Fe-OH

for the P450-catalyzed

carboxaldehyde.

AND

PROSPECTS

1. Examples of regulatory mechanisms in P450 expression4

Regulatory

step

Transcription

P450 examples 1A1,

1A2,

2B1,

2B2,

2C7,

2C11,

2C12, 2D9, 2E1, 2H1, 2H2, 3A1/2, 3A6, 4A1, hAl, 11B1, 17, 21A1 Processing mRNA

and stabilization

Translation

and stabilization

enzyme

4Selected

examples,

1A1, 1A2, 2B1, 2B2, 2C12, 2E1, 2H1, 2H2, 3A1/2, 3A6, hAl 2E1,

3A1/2,

from various

3A6

mammalian

species,

are taken from

a recent review (6).

ally activated at birth and in the adult by fasting, whereas the 10-fold increase in the corresponding mRNA accompanying development of the diabetic state is due solely to stabilization (6). Administration of compounds such as ethanol, acetone, and imidazole induces 2E1 protein without affecting mRNA levels, and is probably due to ligand-mediated protection of the enzyme from phosphorylation and degradation (37, 38). Other less common points of P450 regulation occur at the level of mRNA processing and translation, as shown in the summary in Table 1. Investigation of the mechanisms underlying the inductive response of P450 to the administration of xenobiotic compounds has been of considerable value in understanding the role of cytochrome P450 in the potentiation of cellular toxicity and carcinogenicity caused by these substances. In recent years, increasing attention has been given to discerning the molecular mechanisms involved in the physiological regulation of P450 expression. The P450 monooxygenase system appears in the liver of some species soon after birth, and expression of particular P450 forms in neonates is coincident with weaning (39). The mechanisms of neonatal P450 expression are unknown; however, changes in the methylation state of the 2El gene have been linked to transcriptional activation in such animals (40). Activation and suppression of gene transcription are both possible modes of sex-dependent regulation of P450 expression during puberty, which in some cases requires neonatal exposure to hormones, or imprinting (41, 42). The P450 forms responsible for steroid hormone biosynthesis #{128}re regulated by ACTH, which enhances transcriptional activity through a cAMP-dependent mechanism. However, the known steroidogenic P450 genes do not contain sequences that are related to the cAMP-responsive element described for most cAMP-regulated genes. Indeed, each steroidogenic P450 gene may be regulated by a unique element (43). Most of the P450s characterized to date have been obtained from liver tissue, but many of these same forms are also expressed in extrahepatic tissues, although generally to a lesser extent. Some P450 isoforms such as those involved in the biosynthesis of aldosterone and sex hormones are expressed exclusively in steroidogenic tissues (44). Other examples of tissue-specific expression include P450 2G1, which occurs only in olfactory tissue (45, 46) and a prostaglandin w-hydroxylase that is present exclusively in the lung of pregnant animals (47). Some forms of P450 are not uniformly expressed throughout a given tissue, as seen in the heterogeneous expression of 2E1 within liver, which is due to regional differences in transcriptional activity (48).

ci:1111)

[F8O..HA. +

TABLE

deformyla-

671

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PROSPECTS

IN P450

RESEARCH

vigor of investigative work in the by the attendance and presentations at meetings and the original papers and reviews

Considering

the

present

P450 field, as judged

international being published in a wide variety of journals, we may safely predict that advances will come even more rapidly. Biochemists, pharmacologists, and toxicologists were among the first to appreciate the importance of this CO-binding pigment, but with the availability of the purified cytochromes, chemists, biophysicists, and molecular geneticists have brought their expertise to the field in increasing numbers. Improved regulation

basic

knowledge

of enzyme

catalysis

and

For many of us with a basic orientation in research, the most exciting prospect is an understanding of the relationship between structure and function for such a versatile catalyst. For that purpose, the 3-dimensional structure (49) and related properties

(50, 51) of the bacterial,

101) are highly

interesting.

branous

from

P450s

cytosolic

However,

vertebrates

(which

P4SOcam

the structure may

(P450

of mem-

be significantly

different from the bacterial cytochrome) will have to be established by X-ray crystallography, which is no easy task. We have yet to understand why nature has devised a series of catalysts with overlapping activities and low turnover numbers rather than with greater specificity and higher catalytic rates. With respect to reaction mechanisms, the elusive active oxygen needs to be stabilized and characterized; it may be an iron-oxenoid species, as has been postulated, or it may exist as resonance forms in which the sulfur, iron, and oxygen atoms are at various valence states. Much also remains to be learned about regulatory mechanisms. For example, phenobarbital is one of the most widely used inducers, but a receptor for this drug has yet to be identified, and it is not clear why a compound that has been in the environment only in modern times is involved in the induction of an ancient subfamily of P450 cytochromes. Progress in some more applied areas, as discussed below, will be rapid only as our fundamental understanding of this remarkable superfamily of catalysts becomes more sophisticated. Synthesis screening

of fine

chemicals,

including

drug

design

and

-

At recent national and international meetings on drug metabolism, it became clear that the pharmaceutical industry is beginning to benefit greatly by screening potential drugs with liver microsomal preparations and reconstituted P450 enzyme systems. Improved predictions of drug metabolic stability and toxicity will be possible as more cytochromes become available from various organdIes, tissues, and species, particularly from the human. The synthesis of fine chemicals may be aided by the increased availability of purified P450s capable of inserting oxygen atoms or bringing about other reactions that in some cases appear not to follow the rules of organic chemistry. Many of these reactions are both regioand stereospecific and occur, for example, on hydrocarbon chains or other structures far removed from activating groups. The w- or (w-1)-hydroxylation of fatty acyl or alkyl groups is but one of the many reactions effected by P450 that are difficult for the organic chemist to accomplish. With improved knowledge of the active site of P450 and of the role of specific amino acid residues, model (nonprotein) catalysts may be devised that will mimic the

cytochromes in function but have far greater stability. The problem of providing both electrons and oxygen on a continuous basis may be circumvented if alternative oxygen donors can be used in model systems such as iodosobenzenes, peracids, and hydroperoxides. Removal

of undesirable

environmental

chemicals

Most synthetic chemicals are useful and may even be necessary for human health and agricultural productivity in an increasingly overpopulated world, but their persistence in the environment is a cause for concern. Considering that the technology already exists for the heterologous expression of foreign proteins, one could anticipate the large-scale use of individual mammalian P450s in suitable microorganisms to dispose of toxic chemicals in the environment. A wiser approach would be to detoxify chemicals in this manner before their entry into the environment. Biomedical carcinogen,

applications and alcohol

related to steroid, metabolsim.

drug,

Many possibilities come to mind where improved knowledge about P450 cytochromes and their distribution in the human population could be used to prevent or alleviate medical problems, For predictive purposes with respect to the toxicity, mutagenicity, or carcinogenicity of certain drugs and other foreign compounds, we will need to know the quantitative pattern of P450s in each individual. Immunochemical assays may help in reaching this goal. Predictions may then be possible about an individual’s reaction to xenobiotics that might result in adverse effects, or in rare instances be lethal (52). Our present knowledge of the P450 system already indicates that many factors determine P450 levels in an individual, including genetic background, dietary habits, alcohol intake, hormonal levels, and exposure to foreign

compounds

that act as inducers

or repressors.

Assuming

that

one can first decide whether the level of a particular P450 in an individual is at a dangerously high or low level, it will be necessary to devise methods to change that level. Various in-

ducers

and inhibitors

could be used,

and eventually

human

gene therapy may prove useful. Major medical problems that directly involve P450 include congenital adrenal hyperplasia, a relatively frequent and complex human disorder involving, among other factors, a genetic deficiency in steroid 21-hydroxylase (53). Other examples are the toxicities and carcinogenicities associated with alcoholism (54), as well as pathological conditions involving drug overdose, drug interactions, or overexposure to certain other environmental chemicals. Because P450s facilitate detoxification in some instances and in others convert biologically inert compounds to harmful products, it will be a challenge to unravel the complexities of the reactions of P450 cytochromes and related enzymes and to devise safe methods for their regulation.

I!i1

Research in this laboratory was supported by grant DK-h0339 from the National Institutes of Health and grant AA-0622h from the National Institute on Alcohol Abuse and Alcoholism. REFERENCES 1. Lu,

A. V. H.,

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