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Archives of Biochemistry and BiophysicsVol. 396, No. 1, December 1, pp. 16–24, 2001doi:10.1006/abbi.2001.2585, available online at http://www.idealibrary.com on

Expression, Purification, and Biochemical Characterizationof A Human Cytochrome P450 CYP2D6-NADPHCytochrome P450 Reductase Fusion Protein

Yusuf Y. Deeni,* Mark J. I. Paine,* Andrew D. Ayrton,† Stephen E. Clarke,†Richard Chenery,† and C. Roland Wolf*,1

*Biomedical Research Centre, Ninewells Hospital & Medical School, University of Dundee, Dundee,DD1 9SY, United Kingdom; and †Department of Drug Metabolism and Pharmacokinetics,GlaxoSmithkline Pharmaceuticals, The Frythe, Welwyn, AL6 9AR United Kingdom

Received July 27, 2001, and in revised form August 29, 2001; published online November 9, 2001

Cytochrome P450 CYP2D6 metabolizes a wide range ofpharmaceutical compounds. A CYP2D6 fusion enzyme(CYP2D6F), containing an amino-terminal humanCYP2D6 sequence and a carboxyterminal humanNADPH-cytochrome P450 oxidoreductase (CPR) moiety,was constructed. High levels of expression wereachieved in Escherichia coli (60–100 nmol/liter) and theenzyme was catalytically active with optimal activitiesachieved in the presence of the antioxidant, GSH. Turn-over values for bufuralol 1*-hydroxylation, metoprolola-hydroxylation, O-desmethylation, and dextromethor-phan O-demethylation, using membranes expressing thefusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min21, respec-tively. These values were similar to E. coli membraneswhich coexpressed human CYP2D6 and CPR (CYP2D6/R). The Km and kcat values for bufuralol metabolism wereestimated to be 10.2 mM and 4.1 min21, respectively. Thenzyme was purified using ion-exchange chromatogra-hy, affinity chromatography (2*-5* ADP-Sepharose),nd gel filtration. Estimated turnover rates for bufuralol*-hydroxylation, metoprolol a-hydroxylation, O-des-

methylation, and dextromethorphan O-demethylationwere 1.2, 0.52, 0.79, and 0.76 min21, respectively. Bu-furalol 1*-hydroxylase activity by purified CYP2D6F wasenhanced by phospholipids and added CPR. TheCYP2D6F enzyme was able to stimulate CYP3A4 testos-terone 6b-hydroxylase activity in a reconstitution sys-em indicating that electron transfer may be largely in-ermolecular. The catalytically self-sufficient CYP2D6Fnzyme will facilitate investigations of P450-CPR inter-ctions and the development of new biocatalysts.

© 2001 Elsevier Science

1

To whom correspondence should be addressed. Fax: 144 (0)1382668278. E-mail: rooney@dundee.ac.uk.

16

Key Words: CYP2D6; drug metabolism; cytochromeP450; NADPH cytochrome P450 reductase; fusion;purification.

Cytochrome P450s2 catalyze the metabolism of anextremely wide range of xenobiotic compounds such asdrugs, carcinogens, and toxins (1, 2). They also catalyzethe biosynthesis of endogenous compounds such as ste-roids, vitamins, biogenic agents, and leukotrienes (2).Cytochrome P450 CYP2D6 is of particular medical in-terest because of its major role in the metabolism of alarge number of widely used therapeutic agents suchas cardiovascular drugs, b-adrenergic blocking agents,and tricyclic antidepressants (3). Between 5 and 10% ofthe Caucasian population are unable to metabolizeCYP2D6 substrates due to inactivation of the CYP2D6gene (4) and are therefore more susceptible to adversedrug reactions (5–7).

The substrate specificity of CYP2D6 has been exten-sively characterized. Most CYP2D6 substrates containa basic nitrogen atom, which is thought to interact witha negatively charged atom in the active site (8–10).Current evidence based on site-directed mutagenesisstudies (11, 12), NMR analysis, and homology model-

2 Abbreviations used: P450 or CYP, cytochrome P450; CPR,NADPH-cytochrome P450 reductase; EDTA, ethylenediaminetet-raacetic acid; TSE, Tris/sucrose EDTA buffer; DTT, dithiothreitol;SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electro-phoresis; PMSF, phenylmethylsulfonyl fluoride; HPLC, high-pres-sure liquid chromatography; GSH, reduced glutathione; CYP2D6F,CYP2D6 fused with human NADPH-cytochrome P450 reductase;CYP2D6/R, CYP2D6 coexpressed with CPR; TB, Terrific broth;

Chaps, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate.

0003-9861/01 $35.00© 2001 Elsevier Science

All rights reserved.

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17CYP2D6 FUSION

ing work (13) suggests that the active-site residue in-volved is the acidic residue Asp301.

Substrate metabolism by a cytochrome P450 is de-pendent on the supply of reducing equivalents from itsredox partner NADPH cytochrome P450 reductase(CPR). CPR is a 78-kDa membrane-bound flavoprotein,containing discrete FMN, FAD, and NADPH bindingdomains. Interactions between the two enzymes occuron the cytoplasmic surface of the endoplasmic reticu-lum where electrons are transferred from NADPH toP450 via FAD and FMN cofactors. The determinants inthe redox protein interactions and the formation of aproductive monooxygenase complex are as yet notclearly defined. However, electrostatic, (14), hydropho-bic, and van der Waals forces (15) have all been impli-cated. CPR may also be naturally fused to P450, as inthe case of the prokaryotic enzyme in Bacillus megate-rium P450-BM3 (16), or to other heme binding en-zymes such as the nitric oxide synthases (17, 18). P450-BM3 catalyzes myristate v-2-hydroxylation with aturnover rate of over 1500 min21, which is among thefastest of any known P450-catalyzed reaction. An arti-ficially constructed fusion between rat P450 CYP1A1and rat CPR was originally constructed by Murakamiet al. (19). Since then, several fusions with rat CPRhave been constructed including bovine CYP17a (20),human CYP3A4 (21), human CYP1A1 (22), CYP4A1(23), and rat CYP2C11 (24). In addition, yeast CPR hasbeen fused to rat CYP1A1 (25) and human CYP3A4(26).

The P450 enzyme family plays a major role in drugmetabolism and is therefore of wide interest in bothacademic and commercial areas of research. Catalyti-cally self-sufficient P450-CPR fusion enzymes are ex-tremely useful toward probing the biochemical mecha-nisms involved in catalysis and electron transfer be-tween P450 and CPR. They also have importantpractical applications in the development of efficientP450-based biocatalysts and high throughput screen-ing systems. In this paper, we report on the expression,purification, and biochemical characterization of a re-combinant fusion protein containing the medically im-portant human CYP2D6 fused with human CPR. Toour knowledge, this is the first report of a functionalycomplete human P450 fusion enzyme system. We showthat the CPR moiety provides catalytic self-sufficiencyand facilitates purification through affinity chromatog-raphy with 29-59-ADP-Sepharose. Such an enzyme willbe extremely useful for functional studies. The fusionsystem also has a number of biotechnological applica-tions including as a bioreactor for the synthesis ofregio- and stereoselective pharmaceutical compoundsand also in the high throughput screening of novel

drug compounds. j

MATERIALS AND METHODS

Materials. Restriction enzymes were purchased from Gibco LifeTechnologies, Inc.; [g35-S]dATP was from Amersham Pharmacia Bio-ech. Unless stated otherwise, all other enzymes and reagents wereurchased from Sigma (Poole, Dorset, UK).Construction of human P450 CYP2D6:human NADPH-P450 re-

uctase fusion plasmid. The strategy for constructing theYP2D6F enzyme is shown in Fig. 1. The plasmid pB42 containsuman CYP2D6 cDNA fused with an N-terminal omp A leaderequence in the pCWori1 expression vector as described (27). Plas-id pJR4 contains full-length human CPR cDNA (derived from auman skin fibroblast cDNA library (28)) cloned into the pCWori1ector as described (29). Plasmid pB42 was digested with SphI andbaI and gel-purified, to obtain a vector/CYP2D6 fragment of ;6.5

kb. PCR amplification using PLATINUM pfx DNA polymerase(Gibco BRL) was carried out across the SphI site at position 1328 inCYP2D6 using a forward primer to nucleotides 1119–1139 (MP4;59-TATGACCCACATGACATCCCG-39), and a reverse primer to nu-cleotides 1470–1491 (LK4; 59-GGAGGTCAATGTCTGAATTTTGTC-GACGCGGGGCACAGCACAAAGCTC-39). LK4 contains an ApoI re-triction enzyme sequences (underlined) as well as CPR nucleotides62–282. These sequences replace the C-terminal CYP2D6 stopodon with a six nucleotide linker sequence coding for Val Asp, thus

FIG. 1. Strategy used to engineer the human CYP2D6 and humanNADPH-P450 reductase fusion protein (CYP2D6F). Experimentaldetails are described under Materials and Methods. PtacPtac, tan-dem tac promoter.

oining the P450 to CPR. The PCR-generated fragment was digested

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18 DEENI ET AL.

with SphI and ApoI and gel purified, to obtain the ;175-bp linkerfragment, and pJR4 was digested with ApoI and XbaI to obtain a;1.9-kb CPR fragment. These fragments were ligated to the ;6.5 kbpCW/CYP2D to generate pYD2b (Fig. 1). The construct expresses thechimeric CYP2D6-CPR fusion enzyme, CYP2D6F. The authenticityof the regions generated by PCR was verified by sequencing (30).

Preparation of membrane fractions of E. coli. Plasmids weretransformed into E. coli DH5a cells. Overnight cultures (10 ml)rown in Luria-Bertani broth with ampicillin (50 mg/ml) selection at7°C were used to inoculate a 1-liter culture of Terrific broth (TB)ontaining 100 mg/ml ampicillin, 1 mg/ml thiamine and trace ele-

ments. Cultures were grown at 37°C and 230 rpm to an OD600 ofbetween 0.6 and 1.0. Following the addition of isopropyl-1-thio-b-D-alactopyranoside (1 mM) and d-aminolevulinic acid (0.5 mM), cul-

tures were grown at 26°C and 190 rpm for 40–48 h. Cells were thenharvested by centrifugation and resuspended in 1/20 of the originalculture volume with ice-cold TSE (50 mM Tris-acetate, 250 mMsucrose, 0.5 mM EDTA, pH 7.6). Lysozyme was added to 0.25 mg/mland cells were incubated at 4°C with gentle shaking to producespheroplasts. The spheroplasts were harvested by centrifugation for15 min at 2800g and 4°C. The pellet was resuspended in 1/40 of theoriginal culture volume with ice-cold 100 mM potassium phosphatebuffer (pH 7.6), containing 6 mM magnesium acetate, 20% glycerol,0.1 mM dithiothreitol, 1 mM PMSF, 0.25 mg/ml protease inhibitorcocktail (Sigma). To prepare membrane fractions, spheroplasts werelysed by sonication with several short bursts (10 s) at full powerusing Soniprep 150 (MSE Scientific Instruments, Crawley, UK).Following a clearing spin at 10,000g, 15 min, 4°C, the supernatantwas ultracentrifuged at 100,000g (Sorvall Ultra Pro 80 with A641rotor). The membrane pellet was resuspended, using a Dounce ho-mogenizer, in 20 mM potassium phosphate buffer (pH 7.5), contain-ing 20% glycerol, 0.5 mM EDTA, 0.1 mM dithiothreitol, 1 mM PMSF,0.25 mg/ml protease inhibitor cocktail.

Purification of CYP2D6 fusion. E. coli membranes expressingfusion enzyme were solubilized by mixing at 4°C for 60 min in 2%Emulgen 911 (Kao, Chemical Division, Tokyo, Japan) and ultracen-trifuged at 100,000g. The supernatant fraction was passed over a5-ml Mono Q Hi-Trap column (Pharmacia), preequilibrated with 50mM potassium phosphate buffer (pH 7.5) containing 20% glycerol,0.1% Emulgen 911, 0.5 mM EDTA, 0.1 mM DTT, 1 mM PMSF, 0.25mg/ml protease inhibitors cocktail, and 10 mM FMN. The column waswashed with 10 column vol of equilibration buffer. Column-boundproteins were eluted in 40-ml step gradients of equilibration buffercontaining 0.1, 0.2, 0.3, and 0.4 M KCl. The fusion protein elutedwith the 0.3 to 0.4 M NaCl fractions and these were pooled andloaded onto a 5 ml 29-59-ADP-Sepharose (Sigma) column, equili-brated in affinity buffer (20 mM potassium phosphate, pH 7.7, 250mM KCl, 20% glycerol, 0.1% Emulgen 911, 0.5 mM EDTA, 0.1 mMdithiothreitol, 1 mM PMSF, 0.25 mg/ml protease inhibitor cocktail,and 10 mM FMN). The column was washed with 50 ml of affinityuffer and the fusion protein was eluted with affinity buffer contain-ng 10 mM 29-AMP and subjected to size-exclusion chromatographysing a Sephacryl S-200 HR column (Pharmacia) to remove lowerolecular weight degradation products. Briefly, the column was

quilibrated with 20 mM phosphate buffer (pH 7.5) containing 20%lycerol, 250 mM KCl, 0.1% Emulgen 911, 0.5 mM EDTA, 0.1 mMithiothreitol, and 1 mM PMSF. About 4 ml of the sample waspplied to the column and 0.5 ml fractions were collected. The frac-ions containing homogeneous fusion protein, as judged by SDS-AGE and Coomassie brilliant blue R staining, were pooled and thenoncentrated to about 1 ml following 29-59-ADP affinity purification.riefly, around 20–30 ml of pooled fractions was applied to a 0.5 ml9-59-ADP-Sepharose column, washed with affinity buffer, andluted with 1 ml affinity buffer containing 10 mM 29-59-AMP as

described above. Samples were then dialyzed at 4°C against 20 mMphosphate buffer (pH 7.4) containing 20% glycerol, 0.5 mM EDTA,

0.1 mM DTT for 24 h, and stored in small aliquots at 270°C.

Spectroscopy, flavin determination, and activity assays. Visibleabsorption spectra were recorded, at ambient temperature, using aShimadzu MPS-2000 spectrophotometer (Shimadzu, Kyoto, Japan).P450 content was determined essentially according to the methods ofOmura and Sato (31). Assays for NADPH-cytochrome c reductionwere carried out in 0.3 M potassium phosphate buffer, pH 7.7, atambient temperature as described (28, 32). The FMN and FADcontent was determined by HPLC analysis as previously described(33).

Assays for (6) bufuralol and (6) metoprolol metabolisms werecarried out as described before (27, 34, 35), with minor modification,using 20 pmol of P450 (CYP2D6F or 2D6/R) and an incubation timeof 10 min. The final concentrations of bufuralol and metoprolol were20 and 40 mM, respectively. For kinetic analysis of bufuralol metab-lism, the range of substrate concentration used was 0–50 mM.extromethorphan O-demethylase assays were carried out at 37°C

n 50 mM potassium phosphate buffer, pH 7.4, containing 100 mMdextromethorphan, 20 pmol of CYP2D6 or CYP2D6F, and anNADPH-generating system (5 mM glucose 6-phosphate, 1 U glucose-6-phosphate dehydrogenase, 1 mM NADP) in a total volume of 200ml. Reactions were initiated by the addition of the NADPH-generat-ing system after preincubation for 3 min at 37°C. The reactions werestopped after 10 min by the addition of 100 ml of 3% (v/v) perchloricacid in ice-cold methanol, placed on ice for at least 5 min, andcentrifuged (12,000g) for 5 min. The supernatant was analyzed byHPLC as described (36) with minor modification. Metabolites wereseparated at 30°C on a Hypersil BDS-C18 (5 mm) 4 3 250-mmcolumn (Agilent Technologies, Stockport, UK) using a gradient ofaqueous ammonium acetate (0.1 M, pH 5) and acetonitrile at a flowrate of 1 ml/min. Metabolite peaks were monitored at lex 270 and lem

312 and the concentration of dextrophan (O-demethyldextromethor-phan) determined by reference to a known standard.

For purified recombinant CYP2D6F and CYP2D6, the assays forbufuralol, metoprolol, and dextromethorphan metabolisms were car-ried out as with membranes, using a described reconstitution proce-dure (37). Briefly, purified recombinant CYP2D6F or CYP2D6 (50pmol) was mixed with 2 ml of a solution of dilauroylphosphatidylcho-line and L-a-dioleoylphosphatidylcholine (1:1; 10 mg/ml) and, whereindicated, 50–400 pmol of purified recombinant human CPR, toobtain P450 to CPR ratios between 1:1 and 1:8. To reconstitute theenzymes, samples were incubated at 37°C for 5 min and then at roomtemperature for 10 min. Bufuralol, metoprolol, or dextromethorphanwas added to a final concentration of 20, 40, or 100 mM, respectively.

he mixtures were diluted to 80 ml by the addition of 50 mM potas-ium phosphate buffer (pH 7.4), containing 3 mM GSH, and prein-ubated for 3 min at 37°C. Reactions were initiated by the addition of0 ml of 53 NADPH-generating system (25 mM glucose 6-phosphate,U glucose-6-phosphate dehydrogenase, 5 mM NADP) in 50 mM

otassium phosphate buffer, pH 7.4) and allowed to proceed at 37°Cor 10 min. Adding 50 ml of 3% perchloric acid in ice-cold methanoltopped bufuralol and metoprolol reactions; dextromethorphan as-ays were stopped with 2.5 ml 60% perchloric acid. Metabolites were

analyzed by HPLC as described elsewhere. A concentration range of0–150 mM bufuralol was used for the kinetics on bufuralol metabo-lism by the purified fusion protein. The determination of 6b-hy-

roxytestosterone (testosterone metabolism) by HPLC was as de-cribed before (34). Reaction mixtures contained 200 mM testoster-

one. All determinations were performed in triplicate, and apparentkinetic parameters were calculated using the computer sharewarepackage Hyper.exe, Version 1.1s at http://www.liv.ac.uk/;jse/abouthyp.html.

Other procedures and assays. Proteins were separated by SDS-PAGE on 8% acrylamide gels (38) and visualized by staining withCoomassie blue R. For Western blot analysis, proteins separated bySDS-PAGE gel were transferred onto Hybond-ECL nitrocellulosemembrane (Amersham) (39) and probed with rabbit anti-human

CPR as previously described (27). Protein content was estimated by

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19CYP2D6 FUSION

the Bradford method using bovine albumin as the standard (40).Deoxynucleotide sequencing was carried out by the method of Sangeret al. (30).

RESULTS

Expression and Characterization of CYP2D6F

E. coli transformed with CYP2D6F cDNA expressedrecombinant P450 at levels of between 60 and 120nmol/liter of culture. Membranes prepared followinghigh-speed centrifugation contained around 0.06–0.09nmol of P450 per mg of protein and had cytochrome creductase activities in the range 0.3–0.4 mmol/min/mgf protein. A protein of approximately 129 kDa wasisible following SDS-PAGE of E. coli membranes ex-ressing CYP2D6F (Fig. 2A, lane 2). The size is con-istent with the molecular mass predicted from theDNA sequence, and the polypeptide reacted with rab-it anti human CPR (Fig. 2B, lane 2). Similar resultsere obtained when the CYP2D6F polypeptide was

FIG. 2. SDS-PAGE (A) and Western blot analysis (B) of CYP2D6Fexpressed in E. coli. Membrane preparations from E. coli expressingcontrol plasmid pCW (lane 1) or expressing recombinant CYP2D6F(lane 2) were solubilized in sample loading buffer (31 mM Tris-HCl,pH 6.8, 2% SDS, 10% glycerol, 100 mM 2-mercaptoethanol, 0.002%bromphenol blue). Approximately 10 mg of total protein was resolvedby 8% SDS-PAGE (38) and stained with Coomassie brilliant blueR-250. Proteins were transferred to nitrocellulose (39) and probedwith human CPR antibodies. The bands indicated by arrows corre-spond to the fusion polypeptide. Size markers are shown on the left.

TA

Specific Activities of Recombinant CYP2

1-OH-bufuralol a-OH-metoprolo

2GSH 1GSH 2GSH 1G

CYP2D6F 3.20 6 0.30 5.60 6 0.80 0.20 6 0.01 0.40 6CYP2D6/R 5.75 6 1.12 7.50 6 1.20 0.32 6 0.01 0.30 6

a Assays were performed as described under Materials and Metho20 mM; metoprolol (6), 40 mM; dextromethorphan, 100 mM. Each valu

expressed nmol/min/nmol P450.

probed with CYP2D6 antibodies (data not shown). Ofnote is the fact that degradation products were visibleas lower molecular weight bands in the Western blot,indicating that the fusion protein is prone to proteoly-sis and somewhat unstable.

To determine whether the expressed recombinantCYP2D6F protein was catalytically active, theCYP2D6F membrane fraction was incubated with theCYP2D6 substrates bufuralol, metoprolol, and dextro-methorphan. Assays were carried out in the presenceand absence of the antioxidant GSH, which has beenshown to enhance some P450-mediated reactions (41–44). Also, comparisons were made with E. coli mem-branes which coexpressed CYP2D6 and human CPR asseparate proteins (CYP2D6/R). As shown in Table I,CYP2D6F was catalytically active and substrate turn-overs were increased by around 2-fold in the presenceof the antioxidant, GSH. Under these conditions, bu-furalol 19-hydroxylation by the CYP2D6F was slightlylower than that by CYP2D6/R (0.25-fold). However,dextromethorphan O-demethylation was higher (1.6-fold) as was metoprolol a-hydroxylation (1.33-fold) andO-demethylation (1.2-fold). Metoprolol can be attackedat two different positions by CYP2D6 to form O-des-methylmetoprolol and a-hydroxymetoprolol (45). Theratio of O-desmethylmetoprolol:a-hydroxymetoprololformed by the fusion enzyme was 2, and the same asthe coexpressed enzymes. This is similar to the ratio of2–2.5 reported for CYP2D6 expressed in bacteria (27)and insect cells (35).

The kinetic properties of the CYP2D6F enzyme for(6)-bufuralol hydroxylation are summarized in TableII. The fusion protein exhibited very similar kineticproperties to the holo CYP2D6 enzyme with an appar-ent Km value of 10.2 mM. This was similar to the valueof 10.7 mM obtained with the coexpression system, andin the range 5–60 mM reported for other preparationsof CYP2D6 enzymes (36, 37, 46). The kcat for CYP2D6Fof 4.1 min21, which was a little lower than the value of5.8 min21 for CYP2D6/R, was also similar to some ofthe reported values in the literature (37, 46, 47).

I

F and CYP2D6/R in E. coli Membranesa

ODM-metoprolol ODM-dextromethorphan

2GSH 1GSH 2GSH 1GSH

.01 0.40 6 0.01 0.72 6 0.01 3.19 6 0.68 6.19 6 0.77

.01 0.61 6 0.02 0.60 6 0.01 2.38 6 0.39 3.93 6 0.85

The final concentrations of the substrates used were bufuralol (6),epresents the mean 6 SD of triplicate determinations. Activities are

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20 DEENI ET AL.

Purification of CYP2D6 Fusion Protein

In order to study the isolated fusion protein, a 3-stepseparation procedure was employed. Mono-Q anion-exchange chromatography was used as a preliminarystep to remove around 30–50% of the endogenous bac-terial proteins. This was followed by an affinity-purifi-cation step using 29-59-ADP-Sepharose 4B which bindso the flavoprotein domain. The fusion enzyme wasusceptible to proteolytic cleavage and therefore, a fi-al gel-filtration step over Sephacryl S-200 HR wassed to separate full-length CYP2D6F enzyme fromhe cleaved P450 and reductase domains. The finalurified product is shown in Fig. 3. Yields of around0–15 nmol of pure fusion enzyme were obtained periter of E. coli culture.

Flavin analysis indicated that purified CYP2D6Frotein contained a stoichiometric 1:1 ratio of FMN:AD. The FAD content was 7.7 nmol/mg protein and

TABLE II

Kinetic Analysis of 19-Bufuralol Hydroxylation byMembrane Preparations of Recombinant

CYP2D6F and CYP2D6/Ra

kcat (min21) Km (mM) kcat/Km

CYP2D6F 4.1 6 0.3 10.2 6 1.5 0.4YP2D6/R 5.8 6 0.8 10.7 6 2.0 0.5

a Assays and analysis are described under Materials and Methods.ach value represents the mean 6 SD of three determinations.

FIG. 3. SDS-PAGE analysis of purified recombinant CYP2D6Fprotein. Purified recombinant CYP2D6F (lane 1) and recombinantCYP2D6 (lane 2) were resolved by 8% SDS-PAGE as in Fig. 2. Eachlane contains approximately 5 mg protein. Size markers are shown

on the left.

he NADPH-cytochrome c reductase activity was 17.2mmol/min/mg protein. The absorption spectrum of theenzyme is shown in Fig. 4. The oxidized enzyme waslow spin and had an absorbance maximum at 418 nm.There was a shoulder at around 480 nm and weakerpeaks at 538 and 570 nm from the alpha and beta hemebands. Addition of sodium dithionite and carbon mon-oxide gas resulted in the formation of an absorbancepeak at 445 nm. There was a concomitant bleaching ofthe flavin shoulder and a spectral change in the 500–600 nm range resulting in a single peak at about 550nm. The overall spectral characteristics are similar tothose reported for other CYP450-reductase fusions (20,22–24, 48) and consistent with a protein containingboth heme and flavin domains. The difference spectro-photometry estimated a P450 content of about 7.3nmol/mg of the purified fusion protein. This corre-sponds closely to the figure of approximately 7.8nmol/mg protein estimated from the molecular mass(129 kDa) of the protein and to the FAD and FMNcontent (7.7 nmol/mg). Therefore, there was very littleapoenzyme present in the preparation.

Characteristics of Purified CYP2D6F

In order to optimize the conditions for the catalyticactivities of purified CYP2D6F, we investigated theeffects of added Chaps and phospholipids on 19-bu-furalol hyroxylase activity. Bufuralol 19-hydroxylaseactivity was stimulated approximately 3-fold in thepresence of phospholipids (Table III). Chaps had amarginal stimulatory effect (15%) on catalytic activity.The catalytic activities of CYP2D6F are shown in TableIV. These assays were carried out in the presence of aNADPH-generating system, phospholipdids, GSH, and

FIG. 4. Absorbance spectra of 0.32 mM purified recombinantYP2D6F (Fe31) and its reduced CO complex (Fe21-CO) in 50 mMotassium phosphate buffer (pH 7.4) containing 20% glycerol (v/v),.5 mM EDTA, and 0.1 mM DTT.

Chaps. Rates of metoprolol metabolism were roughly

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21CYP2D6 FUSION

similar to those obtained with membrane prepara-tions. However, turnover numbers for bufuralol anddextromethorphan were 4.7- and 8-fold lower thanthose obtained with membrane preparations (Table I).The ratio of O-desmethylmetoprolol:a-hydroxymeto-

rolol formed by the purified fusion enzyme was 1.5,ence slightly lower than the ratio of 2 obtained withembrane preparations.The kinetic properties of the purified CYP2D6F en-

yme for bufuralol 19-hydroxylation showed the fusionrotein to have an apparent Km value of 8 mM. This was

similar to the value of 10.2 mM obtained with themembrane preparations. The kcat of 2 min21 for purifiedCYP2D6F was half the value of 4.1 min21 recorded

ith the membrane preparations.The addition of purified recombinant human CPR to

he 19-bufuralol hydroxylase reaction mixtures alsoignificantly increased catalytic activity. A 2-fold in-rease in bufuralol 19-hydroxylase activity waschieved with a CYP2D6F:CPR ratio of 1:4, which wasimilar to the peak values obtained with CYP2D6hen reconstituted with CPR at 1:6 (Fig. 5). Compar-

son of the catalytic activity of CYP2D6F (1.2 min21)

TABLE III

Effect of Chaps and Phospholipids on the Catalytic Activityof Purified Recombinant CYP2D6F

Reaction conditionsa

Turnover (min21)(Bufuralol 19-hydroxylase)Chaps Phospholipids

2 2 0.27 6 0.021 2 0.31 6 0.032 1 0.92 6 0.041 1 1.07 6 0.06

a Sample reactions containing 50 pmol of purified CYP2D6F wereerformed in the presence or absence of 20 mg of 1:1 mixture of

L-a-dilaurylphosphatidylcholine and L-a-dioleoylphosphatidylcho-line and/or 0.02% Chaps as described under Materials and Methods.Each value is the mean 6 SD of triplicate determinations.

TABLE IV

Specific Activities of Purified Recombinant CYP2D6Fa

Substrate ProductTurnover(min21)

Bufuralol (20 mM) 1-OH-bufuralol 1.20 6 0.12etoprolol (40 mM) a-OH-metoprolol 0.52 6 0.03

ODM-metoprolol 0.79 6 0.05extromethorphan (100 mM) ODM-dextrometorphan 0.76 6 0.01

a The assays and analysis are as described under Materials andethods. Each value is the mean 6 SD of triplicate determinations.he numbers in parentheses indicate the final concentration of sub-

trates used.

with CYP2D6 reconstituted with an equivalent 1:1 mo-lar ratio of CPR (0.13 min21) (Fig. 5) shows the fusedenzyme to be around 10-fold more active.

The above data, which show that CYP2D6F bu-furalol hydroxylase activity is significantly increasedby the addition of phospholipids and reductase, raisedthe possibility that intermolecular interaction is thepredominant route of electron transfer from the CPR tothe P450 domain in CYP2D6F. To investigate this fur-ther, we determined whether CYP2D6F could couplewith another P450 to form a catalytic unit. To test forreconstitution, the human CYP3A4 was incorporatedinto mixtures containing CYP2D6F (1:1 molar ratio),testosterone (a prototypical CYP3A4 substrate), andbufuralol. As shown in Fig. 6, both substrates weremetabolized by the CYP3A4/CYP2D6F complex. The

FIG. 5. Effect of purified human NADPH-P450 reductase on thecatalytic activity of recombinant CYP2D6 and CYP2D6F. Samplereactions containing 50 pmol of purified CYP2D6 or CYP2D6F wereassayed with varying amounts of purified CPR as described underMaterials and Methods. Each point is the mean 6 SD of a triplicatedetermination.

FIG. 6. Coupling of purified recombinant CYP2D6F (withCYP3A4). Equimolar amounts (50 pmol) of purified CYP3A4,CYP2D6F, and CPR were combined as shown. Bufuralol 19-hydrox-ylase (black column) and testosterone 6b-hydroxylase (white column)activities were assayed as described under Materials and Methods.

Results show the mean 6 SD of triplicate determinations.

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Km

22 DEENI ET AL.

6-b-testosterone hydroxylase turnover was slightlylower (0.49 min21) than that of CYP3A4 reconstitutedwith an equivalent 1:1 molar ratio of wild type CPR(0.6 min21). It was notable that the bufuralol 19-hy-

roxylase activity of the CYP3A4/CYP2D6F mixture0.13 min21) was significantly lower than that of

CYP2D6F incubated without CYP3A4 (0.95 min21).This could be due to competition between CYP2D6 andCYP3A4 for electrons from CPR. This is similar toprevious findings with CYP3A4 and CYP2D6 coex-pressed with CPR in membranes of E. coli (34).

DISCUSSION

A general strategy for the expression of recombinantmammalian P450s in E. coli has been developed in ourlaboratory using the bacterial signal peptide, ompA(29). The strategy has enabled us to create a functionalCYP2D6 monoxygenase complex in E. coli membraneswith the coexpression of human CPR (27). Using thissystem we have generated for the first time a chimerichuman monoxygenase enzyme which contains humanCYP2D6 fused to human CPR. The enzyme, CYP2D6F,reduces cytochrome c and has the capacity to metabo-lize CYP2D6 substrates such as (6) bufuralol, (6)metoprolol, and dextromethorphan. Since the N-termi-nal hydrophobic segment of the CYP2D6 domain isintact, the fusion enzyme remains associated with theE. coli membrane fraction following cell lysis and frac-tionation. The membrane content of 60–100 pmolP450/mg protein is somewhat lower than the 300–500pmol/mg protein achieved with other bacterial ex-pressed CYP2D6 (27). However, the apparent kineticparameters for bufuralol metabolism (Kcat 4.1 min21,

m, 10.2 mM, with membrane preparations; Kcat 2in21; Km 8 mM, with purified enzyme) fell within the

ranges reported in the literature (27, 35–37, 46, 47) fordifferent CYP2D6 enzyme systems. Thus, the enzymeis catalytically similar to CYP2D6 coexpressed withCPR.

Reaction buffer conditions can have a marked effecton the catalytic activity of P450 fusion proteins (48). Inparticular, the addition of MgCl2 and cytochrome b5

has been found to enhance metabolism by other P450s.We found that the addition of MgCl2 (30 mM) had up to30% inhibitory effect on 19-bufuralol hydroxylase activ-ity by both CYP2D6F and CYP2D6/R (data not shown),which was similar to the effect reported for humanCYP1A2 fused with rat CPR (48). The addition of cy-tochrome b5 had no effect on the catalytic activity ofeither purified recombinant CYP2D6 or CYP2D6F(data not shown).

Of most interest was the fact that the activities of thefusion protein in membrane preparations were en-hanced 1.5- to 2-fold by the presence of the antioxidant,

GSH. The reason for this is unclear; however, the ad-

dition of an antioxidant has been shown to enhancemetabolism by other P450 (41–44). It is well knownthat the mechanism of electron transfer from NADPHto P450 is not 100% efficient and that reactive H2O2

species may be produced as a result of uncouplingwhen the substrate is not oxidized. It is possible thatGSH may help sequester H2O2, thereby reducing per-oxidative denaturation of CYP2D6, although we do notyet know the degree of uncoupling in the CYP2D6Fsystem. Alternatively, it has been suggested that GSHmay increase the apparent affinity between P450 andCPR (41).

The fusion enzyme was purified and the effect ofphospholipids and exogenous reductase on catalyticactivity determined. CYP2D6F was catalytically activewithout phospholipids; however, its activity was en-hanced over 3-fold by their addition. The precise mech-anism by which lipids influence the catalytic activity ofP450s is unknown. They may improve the solubility,availability, and accessibility of substrates to the ac-tive site of P450s resulting in greater turnover rates.However, it has also been suggested (23, 49–51) thatthey may provide a vehicle for greater aggregation andorientation of the fusion proteins, thus improving in-teractions between domains responsible for electrontransfer. The fusion protein was approximately 10times more active than CYP2D6 reconstituted with anequivalent 1:1 molar ratio of CPR. However, the bu-furalol hydroxylase activity of purified CYP2D6F wasfurther increased by the addition of purified recombi-nant human CPR (Fig. 5), with an optimal ratio ofreductase to CYP2D6F of around 4:1. This result issimilar to the stimulatory effects of added reductase onfusions involving human CYP3A4 (49), rat CYP4A1(23), or rat CYP2C11 (24). However, it is notable thatadded purified rat CPR does not affect fusion enzymescontaining human CYP1A2 (48), human CYP1A1 (22),and bovine CYP17a (52). The reason is unclear. It maybe that in some cases, where fusions are not optimallycreated, P450 reductase does not dock particularly ef-ficiently to transfer electrons due to structural con-straints. Alternatively, it is possible that different elec-tron transfer mechanisms may operate for differentP450 fusion systems, which may also depend on therate limiting step of the substrate used.

The capacity of CYP2D6F to metabolize bufuralol inthe absence of phospholipids suggests intramolecularcoupling and electron transfer. However, the stimula-tion of the catalytic activity by phospholipids andadded reductase indicates that electron transfer maybe intermolecular. Further evidence for intermolecularelectron transfer is provided by the fact that CYP2D6Fcan efficiently couple and transfer electrons to anothercytochrome P450 such as CYP3A4. In this case, the6b-testosterone hydroxylase activity of the CYP3A4

reconstituted with CYP2D6F was only slightly (15%)

rparsacaFtfPa

1

1

1

1

1

1

11

1

1

2

2

2

2

2

2

2

2

2

2

3

33

3

3

3

3

23CYP2D6 FUSION

lower than the activity observed for CYP3A4 reconsti-tuted with equimolar quantities of CPR; indicatingthat CYP3A4 can still couple with the fusion protein.

Hydrophobic membrane anchor domains are presentin both P450 and CPR that function in tethering theenzymes to the surface of the endoplasmic reticulum.Soluble forms of CPR, in which the membrane anchorhas been removed, are incapable of transferring elec-trons to P450s. Thus, anchoring the enzymes to themembrane surface appears to be the principal factorrequired for correct spatial orientation of the redoxcenters for effective electron transfer. This is furtheremphasized by the fact that CYP2D6F, in which thereductase domain is preceded by a 50-kDa polypeptide,is capable of coupling to other P450s. The normal sizeof the CPR anchor domain is ;6 kDa. Thus, theseesults show clearly that the nature and length of theolypeptide anchoring the reductase to the membranere relatively unimportant for effective coupling;ather it is the fact that the enzyme is in the samepatial plane as the P450 redox partner. Experimentsre currently underway to investigate the physico-hemical mechanisms underlying redox protein inter-ctions and electron flow between CPR and P450s.inally, we are exploring the biotechnological applica-ion of CYP2D6F toward the development of systemsor high throughput screening of drugs metabolized by450s. This has the potential to discover new ligandsnd the possibility to predict drug-drug interactions.

ACKNOWLEDGMENTS

This work was supported by GlaxoSmithKline Pharmaceuticalsand United Kingdom Medical Research Council Grant No.G9203175.

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