N4rneP45O: progress and piedictionsJ COON,’ XINXIN DING, STEVENJ. ALPIN D. N. VAZ

Department of Biological Chemistrc The University of Michigan Medical School, Ann Arbor,

0892-6638/92/0006-669I01 - © FASER 669

ABSTRACT The cytochrome P450 gene superfamily en-codes many isoforms that are unusual in the variety ofchemical reactions catalyzed and the number of substratesattacked. The latter include physiologically important sub-stances such as steroids, eicosanoids, fatty acids, lipidhydroperoxides, retinoids, and other lipid metabolites, andxenobiotics such as drugs, alcohols, procarcinogens, anti-oxidants, organic solvents, anesthetics, dyes, pesticides,odorants, and flavorants. Accordingly, it is not surprisingthat these catalysts have come under intensive study in re-cent years in fields as diverse as biochemistry and molecu-lar biology, endocrinology, pharmacology, toxicology,anesthesiology, nutrition, pathology, and oncology. In thisreview, recent advances in our knowledge of the catalyticproperties, reaction mechanisms, and regulation of expres-sion and activity of the P450 enzymes are briefly summa-rized. In addition, the prospects for research in this fieldare considered, and advances are predicted in four broadareas: improved basic knowledge of enzyme catalysis andregulation; synthesis of fine chemicals, including drug de-sign and screening; removal of undesirable environmen-tal chemicals; and biomedical applications related tosteroid, 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.

Key Wo,th: ytochrome P450 Lrozjines oxygen activation regulationof expression . lipid peroxidation . olefin formation from aldehydes

CYTOCHROME P450 HAS BECOME THE subject of intensiveresearch in recent years in many laboratories for two reasons.An understanding of the remarkable versatility of this familyof enzymes is of interest to those studying biological catalysisfrom a fundamental point of view, and an elucidation of thereactions catalyzed has obvious biomedical relevance for in-testigators in endocrinology pharmacology, toxicology,anesthesiology, nutrition, pathology, oncology, and relatedfields. In this brief review, we have summarized recent ad-vances that have led to our present knowledge of the mul-tiplicity of P450 isoforms, substrates, catalytic mechanisms,and regulatory pathways and have predicted some future de-velopments.

DIVERSITY OF REACTIONS CATALYZED

What is now often called the P450 gene superfamily encodesnumerous enzymes, of which more than 150 have so far beencharacterized. These vary from about 10 to over 90% in se-quence identity and occur in biological sources as diverse asmicroorganisms, plants, and animals. Almost all mam-malian tissues contain one or more of these cytochromes invarious organelles, predominantly in the endoplasmic reticu-lum and mitochondria. Some of the P450 isoforms are fairlyspecific in their choice of substrates (for example, thesteroidogenic cytochromes), but many, and particularly

those in the hepatic endoplasmic reticulum, catalyze a sur-prisingly large number of chemical reactions with an almostunlimited number of biologically occurring and xenobioticcompounds (1-6). In the latter category are synthetic en-vironmental chemicals, now estimated at about 250,000,most of which are potential P450 substrates if not inducersor inhibitors of the individual cytochromes. Examples ofxenobiotics that serve as P450 substrates are drugs (includ-ing antibiotics), procarcinogens, antioxidants, organic sol-vents, anesthetics, dyes, pesticides, alcohols, odorants, andflavorants, and a variety of unusual substances in plants andmicroorganisms, which, despite their biological occurrence,are foreign to animals. Many new drugs and other organiccompounds that will be synthesized in the future can also beexpected to be substrates. The physiologically importantsubstrates include steroids, eicosanoids, fatty acids, lipidhydroperoxides, retinoids, acetone, and acetol.

SYSTEMATIC NOMENCLATURE

When definitive evidence was first obtained by enzyme frac-tionation and characterization for multiple forms of P450(7), it was not known whether different species and tissueswould have similar isoforms. The trivial names assigned byvarious investigators were based on the sources used or onspectral properties, electrophoretic mobility, substrates, orinducers, or in some instances numbers or letters were as-signed in series. Chloroperoxidase and P450s have somephysicochemical and catalytic similarities (8) but have no an-tigenic determinants in common (9). Standard methods ofenzyme nomenclature based on the reactions catalyzedproved to be inadequate because various laboratories werestudying different substrates, and in some cases differentproducts or even different chemical reactions with the samecytochrome. With rapid advances in knowledge aboutP450s, and in particular about their amino acid sequencesdetermined directly or predicted from the correspondingcDNAs, it became clear that a general nomenclature basedon divergent evolution (as judged by structural homology)would be helpful. Thanks to Dr. Daniel W. Nebert, who pro-posed such a system and enlisted others in the effort, anomenclature with the following guidelines has been widelyadopted (10).

Those P450 proteins from all sources with 40% or greatersequence identity are included in the same family, as desig-nated by an Arabic number, and those with greater than55% identity are then included in the same subfamily, as

designated by a capital letter. The individual genes (and geneproducts) are then arbitrarily assigned numbers. There arenow 28 families, of which 11 are predicted to exist in allmammals. As an example, the major phenobarbital-

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

f.70 Vni h anuarv 1992 The FASEB lournal CrrmJ T M

inducible cytochrome in rabbit liver microsomes, originallycalled P4SOLM2 or form 2 (7), has been assigned to family 2and subfamily B, and the gene and the enzyme are designatedCYP2B4 and CYP2B4, respectively. The enzyme may also becalled P450 2B4. The main advantage of the unified nomen-clature is that structurally identical or highly similar P450sare easily recognizable regardless of the sources from whichthey were isolated including the species, tissue, and or-ganelle, or the inducer administered to the animal, or any ofa variety of catalytic activities examined.

Those outside the P450 field may find that designationssuch as CYP2B4, CYP3A1O, and CYP1O5C1 are confusing,but the unusual complexity of this enzyme family requires asystematic nomenclature, and it is to be hoped that investiga-tors will also state the sources, inducers, and catalytic activi-ties so as to aid readers of their publications. The unsuitablealternative was a series of trivial names; for example, LM3a,j, MKj1, Hj, alcohol oxygenase, aniline hydroxylase, N-nitro-sodimethylamine demethylase, etc., as were used for what isnow called P450 2E1. The designation P450 is still used, eventhough it was coined to describe a red pigment of unknownfunction having a reduced CO-difference spectrum with amajor band at about 450 nm (11) and later shown to be in-volved in the oxidation of drugs and steroids (12). Even the

term cytochrome is unsuitable, as in most reactions thesecatalysts function as oxygenases rather than as electron car-riers.

DIVERSITY OF CATALYTIC MECHANISMS

The steps involved in the P450-catalyzed reduction ofmolecular oxygen with incorporation of one oxygen atominto 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 recentreviews (5, 6, 14) have summarized newer findings on sub-strate and peroxide activation, and an insightful review byWhite (15) has emphasized the involvement of free radicals

Figure 1. Overall scheme for mechanism of action of P450. Ferepresents the heme iron atom in the active site, RH a substrate,and ROH the corresponding monooxygenation product. R’ LOOHrepresents a lipid hydroperoxide and RH and LO represent the cor-responding reduction products (alkane and oxoacid, respectively).

XOOH represents a peroxy compound that serves as an alternate

oxygen donor to molecular oxygen.

in the mechanism of action of P450 and other monoox-ygenases. Also shown in the scheme is the release of productsof 02 reduction that are not coupled to substrate hydroxyla-tion, such as superoxide, hydrogen peroxide, and in the4-electron NADPH oxidase reaction, water. The well-knownperoxide shunt, in which a peroxy compound such as analkyl hydroperoxide or peracid donates the oxygen atom forsubstrate hydroxylation with no requirement for molecularoxygen or for NADPH as an electron donor, is also shown.

Much remains to be learned about factors controllingregio- and stereospecificity in P450-catalyzed reactions; theavailability of specifically altered proteins from site-directedmutagenesis will be particularly useful in this respect.Although spectral (16) and EPR analysis (17) have providedevidence for activated oxygen intermediates, chemical andphysical approaches are needed to identify these elusive spe-cies in detail. As reviewed elsewhere (6), some interestingvariations on the reactions shown in Fig. 1 are the proposalof a cage radical mechanism for the rearrangement of aprostaglandin endoperoxide to a prostacyclin and a throm-boxane (18), of radical intermediates in dehydrogenationreactions (14), and of aminium radical intermediates in amineoxidations (19). Ortiz de Montellano and Reich (20) havereviewed the unusual properties that permit P450 to contrib-

ute to the regulation of its own activities; these include com-petitive inhibition by many alternative substrates, some casesof mechanism-based inactivation, and stimulation or inhibi-tion by compounds that serve as effectors.

Also shown in the scheme is the ability of ferrous P450 todonate electrons in a stepwise fashion to bring about reac-tions under anaerobic conditions. Although P450 is widelyrecognized to be an oxygenating catalyst, less emphasis hasbeen placed on its role as a reducing catalyst. Many com-pounds, including dyes, N-oxides, and epoxides undergostepwise 2-electron reduction. Another example we havebeen investigating is the reductive cleavage of xenobiotichydroperoxides and lipid hydroperoxides (shown as R’LOOH in Fig. 1) with hydrocarbon formation (21). For ex-ample, cumyl hydroperoxide yields acetophenone andmethane, and the hydroperoxide derived from linoleic acid(l3-hydroperoxy-9,11-octadecadienoic acid) yields 13-oxo-9,1l-tridecadienoic acid (LO in the scheme) and pentane. Thecleavage reaction is believed to involve stepwise 1-electrontransfer, resulting in homolysis of the peroxide oxygen-oxygen bond and generation of an alkoxy radical, with fi-scission of the latter followed by reduction of the secondary

+ LO radical to the hydrocarbon. The alcohol-inducible form ofliver microsomal cytochrome P450 (form 2E1) is the most ac-tive isozyme examined in this reaction (22). We have sug-gested that P450 2E1, in addition to its known damagingeffects in chemical toxicity and chemical carcinogenesis, mayenhance the reductive cleavage of lipid hydroperoxides witha resultant loss in membrane integrity. The likely impor-tance of P450 2El-dependent lipid peroxidation in vivo afterethanol abuse has also been pointed out by Ekstr#{246}mandIngelman-Sundberg (23).

More recently, we have described the P450-dependentconversion of cyclohexane carboxylaldehyde to cyclohexenewith loss of the aldehyde carbon as formate, as shown on thelower left in Fig. 1 (24). This reaction may be a useful modelfor the demethylation reactions catalyzed by the steroido-genic P450s, aromatase and lanosterol demethylase, inwhich an olefinic product and formate are also formed (25,26). The reaction requires P450, NADPH-cytochrome P450reductase, NADPH, and 02. Externally added H202 is ac-tive with P450 in the deformylation reaction in the absence

CYTOCHROME P450: ADVANCES AND PROSPECTS 671

of NADPH and the reductase. In contrast, iodosobenzene isineffective, indicating that iron oxene is not the oxidant, andm-chloroperbenzoic acid and cumyl hydroperoxide are alsoinactive, which indicates that deformylation by H202 ismechanistically distinct from hydroxylation reactions sup-ported by these oxidants. We have concluded that aperoxyhemiacetal-like adduct may be formed between thesubstrate and molecular oxygen-derived hydrogen peroxide.A role for oxygen-derived peroxide in the P450-catalyzed de-methylation of steroids has been proposed by severalresearch groups (27-32). Many other aldehydes also undergothis cleavage reaction, particularly those with branched car-bon chains such as citronellal, which is found in many essen-tial oils and is widely used as an odorant and flavorant (33).Shown in Fig. 2 is a scheme in which an enzyme-basedperoxyhemiacetal-like intermediate, presumably formed

from the heme iron-bound peroxide and the electrophilic al-dehyde carbonyl group, rearranges to yield the olefin andformic acid by either a concerted or a sequential 13-scissionmechanism.

DIVERSITY OF REGULATORY MECHANISMS

Interest in the regulation of cytochrome P450 originallystemmed from observations that the administration of struc-turally diverse chemicals results in induction, that is, an in-crease in the level of one or more isoforms. Categorizationof these inducers is based on the cytochrome that is increased

in amount (34). Thus, polycyclic aromatic hydrocarbons in-duce P450 IA, whereas phenobarbital, glucocorticoids,ethanol, and clofibrate increase the levels of P450s 2B, 3A,2E, and 4A, respectively, but examples are also known wherea single agent induces two or more cytochromes and whereseveral compounds induce the same cytochrome. The induc-tive response of 1AI to TCDD is mediated by a cytosolicreceptor that binds ‘TCDD and then interacts with regula-tory regions in the 5 flanking region of the gene to activatetranscription, thereby leading to increased accumulation of1A1 mRNA and an increase in the level of 1A1 in the en-doplasmic reticulum (35, 36).

Modulation of the transcriptional activity of the gene isthe most common, but not the only, regulatory mechanismof P450 expression. In fact, transcriptional and diverse post-transcriptional mechanisms have been described for the con-trol of P450 2E1 expression. The 2E1 gene is transcription-

Concerted:

Stepwise:

FeOOf OH

Fe_O)H

- Ht!0H + ci::I:J+ Fe-OH

[F8O..HA.0 I

- HOH + [ H]] - ci:1111)+ Fe-OH

2e.. H4

+ Fe-OH

Figure 2. Proposed mechanism for the P450-catalyzed deformyla-tion of cyclohexane carboxaldehyde.

TABLE 1. Examples of regulatory mechanisms in P450 expression4

Regulatory step P450 examples

Transcription 1A1, 1A2, 2B1, 2B2, 2C7, 2C11,

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

Processing andmRNA stabilization 1A1, 1A2, 2B1, 2B2, 2C12, 2E1,

2H1, 2H2, 3A1/2, 3A6, hAl

Translation and

enzyme stabilization 2E1, 3A1/2, 3A6

4Selected examples, from various mammalian species, are taken froma recent review (6).

ally activated at birth and in the adult by fasting, whereas the10-fold increase in the corresponding mRNA accompanyingdevelopment of the diabetic state is due solely to stabilization(6). Administration of compounds such as ethanol, acetone,and imidazole induces 2E1 protein without affecting mRNAlevels, and is probably due to ligand-mediated protection ofthe enzyme from phosphorylation and degradation (37, 38).Other less common points of P450 regulation occur at thelevel of mRNA processing and translation, as shown in thesummary in Table 1.

Investigation of the mechanisms underlying the inductiveresponse of P450 to the administration of xenobiotic com-pounds has been of considerable value in understanding therole of cytochrome P450 in the potentiation of cellular toxic-ity and carcinogenicity caused by these substances. In recentyears, increasing attention has been given to discerning themolecular mechanisms involved in the physiological regula-tion of P450 expression. The P450 monooxygenase systemappears in the liver of some species soon after birth, and ex-pression of particular P450 forms in neonates is coincidentwith weaning (39). The mechanisms of neonatal P450 ex-pression are unknown; however, changes in the methylationstate of the 2El gene have been linked to transcriptional acti-vation in such animals (40). Activation and suppression ofgene transcription are both possible modes of sex-dependentregulation of P450 expression during puberty, which in somecases requires neonatal exposure to hormones, or imprinting(41, 42). The P450 forms responsible for steroid hormone bio-synthesis #{128}reregulated by ACTH, which enhances transcrip-tional activity through a cAMP-dependent mechanism.However, the known steroidogenic P450 genes do not con-tain sequences that are related to the cAMP-responsive ele-ment described for most cAMP-regulated genes. Indeed,each steroidogenic P450 gene may be regulated by a uniqueelement (43).

Most of the P450s characterized to date have been ob-tained from liver tissue, but many of these same forms arealso expressed in extrahepatic tissues, although generally toa lesser extent. Some P450 isoforms such as those involvedin the biosynthesis of aldosterone and sex hormones are ex-pressed exclusively in steroidogenic tissues (44). Other exam-ples of tissue-specific expression include P450 2G1, which oc-curs only in olfactory tissue (45, 46) and a prostaglandinw-hydroxylase that is present exclusively in the lung of preg-nant animals (47). Some forms of P450 are not uniformly ex-pressed throughout a given tissue, as seen in the heterogene-ous expression of 2E1 within liver, which is due to regionaldifferences in transcriptional activity (48).

672 Vol. 6 January 1992 The FASEB Journal COON El AL.

PROSPECTS IN P450 RESEARCH

Considering the present vigor of investigative work in theP450 field, as judged by the attendance and presentations atinternational meetings and the original papers and reviewsbeing published in a wide variety of journals, we may safelypredict that advances will come even more rapidly. Bio-chemists, pharmacologists, and toxicologists were among thefirst to appreciate the importance of this CO-binding pig-ment, but with the availability of the purified cytochromes,chemists, biophysicists, and molecular geneticists havebrought their expertise to the field in increasing numbers.

Improved basic knowledge of enzyme catalysis andregulation

For many of us with a basic orientation in research, the mostexciting prospect is an understanding of the relationship be-tween structure and function for such a versatile catalyst. Forthat purpose, the 3-dimensional structure (49) and relatedproperties (50, 51) of the bacterial, cytosolic P4SOcam (P450101) are highly interesting. However, the structure of mem-branous P450s from vertebrates (which may be significantlydifferent from the bacterial cytochrome) will have to be es-tablished by X-ray crystallography, which is no easy task. Wehave yet to understand why nature has devised a series ofcatalysts with overlapping activities and low turnover num-bers rather than with greater specificity and higher catalyticrates.

With respect to reaction mechanisms, the elusive activeoxygen needs to be stabilized and characterized; it may bean iron-oxenoid species, as has been postulated, or it may ex-ist as resonance forms in which the sulfur, iron, and oxygenatoms are at various valence states. Much also remains to belearned about regulatory mechanisms. For example,phenobarbital is one of the most widely used inducers, buta receptor for this drug has yet to be identified, and it is notclear why a compound that has been in the environment onlyin modern times is involved in the induction of an ancientsubfamily of P450 cytochromes. Progress in some more ap-plied areas, as discussed below, will be rapid only as our fun-damental understanding of this remarkable superfamily ofcatalysts becomes more sophisticated.

Synthesis of fine chemicals, including drug design andscreening -

At recent national and international meetings on drugmetabolism, it became clear that the pharmaceutical industryis beginning to benefit greatly by screening potential drugswith liver microsomal preparations and reconstituted P450enzyme systems. Improved predictions of drug metabolicstability and toxicity will be possible as more cytochromesbecome available from various organdIes, tissues, and spe-cies, particularly from the human. The synthesis of finechemicals may be aided by the increased availability ofpurified P450s capable of inserting oxygen atoms or bringingabout other reactions that in some cases appear not to followthe rules of organic chemistry. Many of these reactions areboth regio- and stereospecific and occur, for example, onhydrocarbon chains or other structures far removed from ac-tivating groups. The w- or (w-1)-hydroxylation of fatty acylor alkyl groups is but one of the many reactions effected byP450 that are difficult for the organic chemist to accomplish.

With improved knowledge of the active site of P450 andof the role of specific amino acid residues, model (non-protein) catalysts may be devised that will mimic the

cytochromes in function but have far greater stability. Theproblem of providing both electrons and oxygen on a con-tinuous basis may be circumvented if alternative oxygendonors can be used in model systems such as iodosoben-zenes, peracids, and hydroperoxides.

Removal of undesirable environmental chemicals

Most synthetic chemicals are useful and may even be neces-sary for human health and agricultural productivity in an in-creasingly overpopulated world, but their persistence in theenvironment is a cause for concern. Considering that thetechnology already exists for the heterologous expression offoreign proteins, one could anticipate the large-scale use ofindividual mammalian P450s in suitable microorganisms todispose of toxic chemicals in the environment. A wiser ap-proach would be to detoxify chemicals in this manner beforetheir entry into the environment.

Biomedical applications related to steroid, drug,carcinogen, and alcohol metabolsim.

Many possibilities come to mind where improved knowledgeabout P450 cytochromes and their distribution in the humanpopulation could be used to prevent or alleviate medicalproblems, For predictive purposes with respect to the toxic-ity, mutagenicity, or carcinogenicity of certain drugs andother foreign compounds, we will need to know the quantita-tive pattern of P450s in each individual. Immunochemicalassays may help in reaching this goal. Predictions may thenbe possible about an individual’s reaction to xenobiotics thatmight result in adverse effects, or in rare instances be lethal(52). Our present knowledge of the P450 system already in-dicates that many factors determine P450 levels in an in-dividual, including genetic background, dietary habits, alco-hol intake, hormonal levels, and exposure to foreigncompounds that act as inducers or repressors. Assuming thatone can first decide whether the level of a particular P450 inan individual is at a dangerously high or low level, it will benecessary to devise methods to change that level. Various in-

ducers and inhibitors could be used, and eventually humangene therapy may prove useful. Major medical problems thatdirectly involve P450 include congenital adrenal hyperplasia,a relatively frequent and complex human disorder involving,among other factors, a genetic deficiency in steroid21-hydroxylase (53). Other examples are the toxicities andcarcinogenicities associated with alcoholism (54), as well aspathological conditions involving drug overdose, drug inter-actions, or overexposure to certain other environmentalchemicals. Because P450s facilitate detoxification in some in-stances and in others convert biologically inert compounds toharmful products, it will be a challenge to unravel the com-plexities of the reactions of P450 cytochromes and related en-zymes and to devise safe methods for their regulation. I!i1

Research in this laboratory was supported by grant DK-h0339from the National Institutes of Health and grant AA-0622h fromthe National Institute on Alcohol Abuse and Alcoholism.

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