Expression, Purification, and Biochemical Characterization of A Human Cytochrome P450 CYP2D6-NADPH Cytochrome P450 Reductase Fusion Protein

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<ul><li><p>Expr lof A -NCyto t</p><p>Yusuf ,Richa*Biome l, UDD1 9 anGlaxoS n</p><p>Receive line</p><p>Cytopharm(CYP2CYP2DNADPwas cachievenzymachievover va-hydrphan Ofusiontively.whichR). Thestimaenzyme was purified using ion-exchange chromatogra-phy, aand ge1*-hydmethywerefuraloenhanCYP2Dteronetem intermolenzymaction 2001 El</p><p>ey0;ific</p><p>ytoemgs,bios,ochsteartrC2D</p><p>gene (4) and are therefore more susceptible to adverse</p><p>1 To w668278.</p><p>16</p><p>Archives of Biochemistry and BiophysicsVol. 396, No. 1, December 1, pp. 1624, 2001doi:10.1006/abbi.2001.2585, available online at http://www.idealibrary.com onffinity chromatography (2*-5* ADP-Sepharose),l filtration. Estimated turnover rates for bufuralolroxylation, metoprolol a-hydroxylation, O-des-lation, and dextromethorphan O-demethylation1.2, 0.52, 0.79, and 0.76 min21, respectively. Bu-l 1*-hydroxylase activity by purified CYP2D6F wasced by phospholipids and added CPR. The6F enzyme was able to stimulate CYP3A4 testos-6b-hydroxylase activity in a reconstitution sys-</p><p>dicating that electron transfer may be largely in-ecular. The catalytically self-sufficient CYP2D6Fe will facilitate investigations of P450-CPR inter-s and the development of new biocatalysts.sevier Science</p><p>drug reactions (57).The substrate specificity of CYP2D6 has been exten-</p><p>sively characterized. Most CYP2D6 substrates containa basic nitrogen atom, which is thought to interact witha negatively charged atom in the active site (810).Current evidence based on site-directed mutagenesisstudies (11, 12), NMR analysis, and homology model-</p><p>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,ession, Purification, and BiochemicaHuman Cytochrome P450 CYP2D6chrome P450 Reductase Fusion Pro</p><p>Y. Deeni,* Mark J. I. Paine,* Andrew D. Ayrtonrd Chenery, and C. Roland Wolf*,1</p><p>dical Research Centre, Ninewells Hospital &amp; Medical SchooSY, United Kingdom; and Department of Drug Metabolismmithkline Pharmaceuticals, The Frythe, Welwyn, AL6 9AR U</p><p>d July 27, 2001, and in revised form August 29, 2001; published on</p><p>chrome P450 CYP2D6 metabolizes a wide range ofaceutical compounds. A CYP2D6 fusion enzymeD6F), containing an amino-terminal human6 sequence and a carboxyterminal human</p><p>H-cytochrome P450 oxidoreductase (CPR) moiety,onstructed. High levels of expression wereed in Escherichia coli (60100 nmol/liter) and thee was catalytically active with optimal activitiesed in the presence of the antioxidant, GSH. Turn-alues for bufuralol 1*-hydroxylation, metoprololoxylation, O-desmethylation, and dextromethor--demethylation, using membranes expressing theenzyme, were 5.6, 0.4, 0.72, and 6.19 min21, respec-These values were similar to E. coli membranescoexpressed human CYP2D6 and CPR (CYP2D6/</p><p>e Km and kcat values for bufuralol metabolism wereted to be 10.2 mM and 4.1 min21, respectively. The</p><p>KP45pur</p><p>CextrdrutheroidCytterelargas candtheCYPhom correspondence should be addressed. Fax: 144 (0)1382E-mail: rooney@dundee.ac.uk.</p><p>CYP2DCYP2DChaps,CharacterizationADPH</p><p>ein</p><p>Stephen E. Clarke,</p><p>niversity of Dundee, Dundee,d Pharmacokinetics,ited Kingdom</p><p>November 9, 2001</p><p>Words: CYP2D6; drug metabolism; cytochromeNADPH cytochrome P450 reductase; fusion;ation.</p><p>chrome P450s2 catalyze the metabolism of anely wide range of xenobiotic compounds such ascarcinogens, and toxins (1, 2). They also catalyzesynthesis of endogenous compounds such as ste-vitamins, biogenic agents, and leukotrienes (2).rome P450 CYP2D6 is of particular medical in-because of its major role in the metabolism of anumber of widely used therapeutic agents suchdiovascular drugs, b-adrenergic blocking agents,icyclic antidepressants (3). Between 5 and 10% ofaucasian population are unable to metabolize</p><p>6 substrates due to inactivation of the CYP2D66 fused with human NADPH-cytochrome P450 reductase;6/R, CYP2D6 coexpressed with CPR; TB, Terrific broth;3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate.</p><p>0003-9861/01 $35.00 2001 Elsevier Science</p><p>All rights reserved.</p></li><li><p>ing work (13) suggests that the active-site residue in-volved is the acidic residue Asp301.</p><p>Substrate metabolism by a cytochrome P450 is de-penderedox(CPR)contaidomaion thelum wP450 vthe reproduclearlybic, ancated.the cariumzymesBM3turnovfastesficiallyand raet al.have bhuman(23), abeen f(26).</p><p>Themetabacademcally stremenismstweenpractiP450-bing syspurificcombiportanour kncomplthat thand faraphybe extsystemtionsregio-and adrug c</p><p>ER</p><p>aternol</p><p>. Unhasnstase2DanencpJRanr aan</p><p>PCco B2DATGideGCtion282n wng</p><p>. 1.PH</p><p>ilstac</p><p>17CYP2D6 FUSIONnt on the supply of reducing equivalents from itspartner NADPH cytochrome P450 reductase</p><p>. CPR is a 78-kDa membrane-bound flavoprotein,ning discrete FMN, FAD, and NADPH bindingns. Interactions between the two enzymes occurcytoplasmic surface of the endoplasmic reticu-</p><p>here electrons are transferred from NADPH toia FAD and FMN cofactors. The determinants indox protein interactions and the formation of active monooxygenase complex are as yet not</p><p>defined. However, electrostatic, (14), hydropho-d van der Waals forces (15) have all been impli-CPR may also be naturally fused to P450, as inse of the prokaryotic enzyme in Bacillus megate-P450-BM3 (16), or to other heme binding en-such as the nitric oxide synthases (17, 18). P450-catalyzes myristate v-2-hydroxylation with aer rate of over 1500 min21, which is among the</p><p>t of any known P450-catalyzed reaction. An arti-constructed fusion between rat P450 CYP1A1</p><p>t CPR was originally constructed by Murakami(19). Since then, several fusions with rat CPReen constructed including bovine CYP17a (20),</p><p>CYP3A4 (21), human CYP1A1 (22), CYP4A1nd rat CYP2C11 (24). In addition, yeast CPR hasused to rat CYP1A1 (25) and human CYP3A4</p><p>P450 enzyme family plays a major role in drugolism and is therefore of wide interest in both</p><p>ic and commercial areas of research. Catalyti-elf-sufficient P450-CPR fusion enzymes are ex-</p><p>ly useful toward probing the biochemical mecha-involved in catalysis and electron transfer be-P450 and CPR. They also have important</p><p>cal applications in the development of efficientased biocatalysts and high throughput screen-tems. In this paper, we report on the expression,ation, and biochemical characterization of a re-</p><p>nant fusion protein containing the medically im-t human CYP2D6 fused with human CPR. Toowledge, this is the first report of a functionaly</p><p>ete human P450 fusion enzyme system. We showe CPR moiety provides catalytic self-sufficiency</p><p>cilitates purification through affinity chromatog-with 29-59-ADP-Sepharose. Such an enzyme willremely useful for functional studies. The fusion</p><p>also has a number of biotechnological applica-including as a bioreactor for the synthesis ofand stereoselective pharmaceutical compoundslso in the high throughput screening of novelompounds.</p><p>MAT</p><p>MTechtechpurc</p><p>CoductCYPhumsequmidhumvectoXbaIkb.(GibCYP59-TcleotGACstric262codojoini</p><p>FIGNADdetademIALS AND METHODS</p><p>ials. Restriction enzymes were purchased from Gibco Lifeogies, Inc.; [g35-S]dATP was from Amersham Pharmacia Bio-less stated otherwise, all other enzymes and reagents wereed from Sigma (Poole, Dorset, UK).ruction of human P450 CYP2D6:human NADPH-P450 re-</p><p>fusion plasmid. The strategy for constructing the6F enzyme is shown in Fig. 1. The plasmid pB42 containsCYP2D6 cDNA fused with an N-terminal omp A leadere in the pCWori1 expression vector as described (27). Plas-4 contains full-length human CPR cDNA (derived from a</p><p>skin fibroblast cDNA library (28)) cloned into the pCWori1s described (29). Plasmid pB42 was digested with SphI andd gel-purified, to obtain a vector/CYP2D6 fragment of ;6.5</p><p>R amplification using PLATINUM pfx DNA polymeraseRL) was carried out across the SphI site at position 1328 in</p><p>6 using a forward primer to nucleotides 11191139 (MP4;ACCCACATGACATCCCG-39), and a reverse primer to nu-</p><p>s 14701491 (LK4; 59-GGAGGTCAATGTCTGAATTTTGTC-GGGGCACAGCACAAAGCTC-39). LK4 contains an ApoI re-enzyme sequences (underlined) as well as CPR nucleotides</p><p>. These sequences replace the C-terminal CYP2D6 stopith a six nucleotide linker sequence coding for Val Asp, thusthe P450 to CPR. The PCR-generated fragment was digested</p><p>Strategy used to engineer the human CYP2D6 and human-P450 reductase fusion protein (CYP2D6F). Experimental</p><p>are described under Materials and Methods. PtacPtac, tan-promoter.</p></li><li><p>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 thechimeriof the r</p><p>Prepatransforgrown i37C wcontainments.betweengalactoptures wharvestculturesucroseand celspherop15 minoriginalbuffer (0.1 mMcocktaillysed busing SFollowiwas ultrotor). Tmogenizing 20%0.25 mg</p><p>Purififusion eEmulgetrifuged5-ml MomM pot0.1% Emmg/ml pwashedproteinscontainwith thloadedbratedmM KCdithiothand 10buffer aing 10 musing amoleculequilibrglyceroldithiothappliedtions coPAGE aconcentBriefly,29-59-ADeluteddescribephospha0.1 mM</p><p>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 of</p><p>raca</p><p>ienent</p><p>sayiedg 2m</p><p>nd, trom</p><p>mromPHospeacystpedin</p><p>rifuC arat</p><p>nousof 1and) dr pralout(3</p><p>l) wandatein Pmeera</p><p>addmixph</p><p>tedl ofglussiu0 mpedweyze0 mby</p><p>yteedAllticagethyherE oas</p><p>-PAbraas</p><p>18 DEENI ET AL.c CYP2D6-CPR fusion enzyme, CYP2D6F. The authenticityegions generated by PCR was verified by sequencing (30).ration of membrane fractions of E. coli. Plasmids weremed into E. coli DH5a cells. Overnight cultures (10 ml)n Luria-Bertani broth with ampicillin (50 mg/ml) selection atere used to inoculate a 1-liter culture of Terrific broth (TB)ing 100 mg/ml ampicillin, 1 mg/ml thiamine and trace ele-Cultures were grown at 37C and 230 rpm to an OD600 of</p><p>0.6 and 1.0. Following the addition of isopropyl-1-thio-b-D-yranoside (1 mM) and d-aminolevulinic acid (0.5 mM), cul-</p><p>ere grown at 26C and 190 rpm for 4048 h. Cells were thened by centrifugation and resuspended in 1/20 of the originalvolume with ice-cold TSE (50 mM Tris-acetate, 250 mM</p><p>, 0.5 mM EDTA, pH 7.6). Lysozyme was added to 0.25 mg/mlls were incubated at 4C with gentle shaking to producelasts. The spheroplasts were harvested by centrifugation forat 2800g and 4C. The pellet was resuspended in 1/40 of theculture volume with ice-cold 100 mM potassium phosphate</p><p>pH 7.6), containing 6 mM magnesium acetate, 20% glycerol,dithiothreitol, 1 mM PMSF, 0.25 mg/ml protease inhibitor(Sigma). To prepare membrane fractions, spheroplasts were</p><p>y sonication with several short bursts (10 s) at full poweroniprep 150 (MSE Scientific Instruments, Crawley, UK).</p><p>ng a clearing spin at 10,000g, 15 min, 4C, the supernatantracentrifuged at 100,000g (Sorvall Ultra Pro 80 with A641he membrane pellet was resuspended, using a Dounce ho-er, in 20 mM potassium phosphate buffer (pH 7.5), contain-glycerol, 0.5 mM EDTA, 0.1 mM dithiothreitol, 1 mM PMSF,/ml protease inhibitor cocktail.cation of CYP2D6 fusion. E. coli membranes expressingnzyme were solubilized by mixing at 4C for 60 min in 2%n 911 (Kao, Chemical Division, Tokyo, Japan) and ultracen-</p><p>at 100,000g. The supernatant fraction was passed over ano Q Hi-Trap column (Pharmacia), preequilibrated with 50assium phosphate buffer (pH 7.5) containing 20% glycerol,ulgen 911, 0.5 mM EDTA, 0.1 mM DTT, 1 mM PMSF, 0.25</p><p>rotease inhibitors cocktail, and 10 mM FMN. The column waswith 10 column vol of equilibration buffer. Column-boundwere eluted in 40-ml step gradients of equilibration buffer</p><p>ing 0.1, 0.2, 0.3, and 0.4 M KCl. The fusion protein elutede 0.3 to 0.4 M NaCl fractions and these were pooled andonto a 5 ml 29-59-ADP-Sepharose (Sigma) column, equili-in affinity buffer (20 mM potassium phosphate, pH 7.7, 250l, 20% glycerol, 0.1% Emulgen 911, 0.5 mM EDTA, 0.1 mMreitol, 1 mM PMSF, 0.25 mg/ml protease inhibitor cocktail,mM FMN). The column was washed with 50 ml of affinitynd the fusion protein was eluted with affinity buffer contain-M 29-AMP and subjected to size-exclusion chromatographySephacryl S-200 HR column (Pharmacia) to remove lower</p><p>ar weight degradation products. Briefly, the column wasated with 20 mM phosphate buffer (pH 7.5) containing 20%, 250 mM KCl, 0.1% Emulgen 911, 0.5 mM EDTA, 0.1 mMreitol, and 1 mM PMSF. About 4 ml of the sample wasto the column and 0.5 ml fractions were collected. The frac-ntaining homogeneous fusion protein, as judged by SDS-nd Coomassie brilliant blue R staining, were pooled and thenrated to about 1 ml following 29-59-ADP affinity purification.around 2030 ml of pooled fractions was applied to a 0.5 mlP-Sepharose column, washed with affinity buffer, and</p><p>with 1 ml affinity buffer containing 10 mM 29-59-AMP asd above. Samples were then dialyzed at 4C against 20 mMte buffer (pH 7.4) containing 20% glycerol, 0.5 mM EDTA,DTT for 24 h, and stored in small aliquots at 270C.</p><p>Omuwereambcont(33).</p><p>Ascarrusinof 1020 aolismDextin 50dextNAD6-phml. Ring sstopacidcentHPLsepacolumaquerate312phan</p><p>FobufurieddurepmolineindicobtaenzytempwasThesiumcuba20 m5 Upotafor 1stopsaysanal015lismdroxscribone.kinepackabou</p><p>OtPAGCoomSDSmemCPRand Sato (31). Assays for NADPH-cytochrome c reductionrried out in 0.3 M potassium phosphate buffer, pH 7.7, att temperature as described (28, 32). The FMN and FADwas determined by HPLC analysis as previously described</p><p>s for (6) bufuralol and (6) metoprolol metabolisms wereout as described before (27, 34, 35), with minor modification,0 pmol of P450 (CYP2D6F or 2D6/R) and an incubation timein. The final concentrations of bufuralol and metoprolol were40 mM, respectively. For kinetic analysis of bufuralol metab-he range of substrate concentration used was 050 mM.ethorphan O-demethylase assays were carried out at 37C</p><p>M potassium phosphate buffer, pH 7.4, containing 100 mMethorphan, 20 pmol of CYP2D6 or CYP2D6F, and an-generating system (5 mM glucose 6-phosphate, 1 U glucose-hate dehydrogenase, 1 mM NADP) in a total volume of 200tions were initiated by the addition of the NADPH-generat-em after preincubation for 3 min at 37C. The reactions wereafter 10 min by the addition of 100 ml of 3% (v/v) perchloricice-cold methanol, placed on ice for at least 5 min, andged (12,000g) for 5 min. The supernatant was analyzed bys described (36) with minor modification. Metabolites were</p><p>ed at 30C on a Hypersil BDS-C18 (5 mm) 4 3 250-mm(Agilent Technologies, Stockport, UK) using a gradient ofammonium acetate (0.1 M, pH 5) and acetonitrile at a flowml/min. Metabolite peaks were monitored at lex 270 and lemthe concentration of dextrophan (O-demethyldextromethor-</p><p>etermined by reference to a known standard.urified recombinant CYP2D6F...</p></li></ul>

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